Ligand of G protein-coupled receptor protein and DNA thereof

ABSTRACT

A GPR7 ligand containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO:1 wherein the N-terminal amino acid residue may optionally be brominated, is useful in developing a receptor-binding assay system with the use of the GPR7 expression system, in screening a candidate compound for a drug such as an antiobestic, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the 35 U.S.C. §371 national stage of PCT applicationNo. PCT/JP02/05915, filed Jun. 13, 2002, which in turn claimed priorityto Japanese patent application no. JP 2001-180562, filed Jun. 14, 2001,JP 2001-216773, filed Jul. 17, 2001, JP 2001-359826, filed Nov. 26,2001, JP 2001-401019, filed Dec. 28, 2001, and JP 2002-154533, filed May28, 2002.

FIELD OF THE INVENTION

The present invention relates to a peptide capable of binding to GPR7,etc., its DNA and use thereof, bovine-derived GPR7 and GPR8, DNAsthereof as well as use thereof.

BACKGROUND ART

Important biological functions including maintenance of homeostasis inthe living body, reproduction, development of individuals, metabolism,growth, control of the nervous, circulatory, immune, digestive ormetabolic system, sensory adaptation, etc. are regulated by cells thatreceive endogenous factors such as various hormones andneurotransmitters or sensory stimulation like light or odor, viaspecific receptors present on cell membranes reserved for theseendogenous factors or stimulation and interact with them. Many of thesereceptors for hormones or neurotransmitters, which take part in suchfunctional regulation, are coupled to guanine nucleotide-bindingproteins (hereinafter, sometimes merely referred to as G proteins), andare characterized by developing a variety of functions through mediationof intracellular signal transduction via activation of the G proteins.In addition, these receptor proteins possess common seven transmembraneregions. Based on the foregoing, these receptors are thus collectivelyreferred to as G protein-coupled receptors or seven transmembranereceptors. As such, it is known that various hormones orneurotransmitters and their receptor proteins are present and interactwith each other to play important roles for regulating the biologicalfunctions. However, it often remains unclear if there are any otherunknown substances (hormones, neurotransmitters, etc.) and receptors tothese substances.

In recent years, accumulated sequence information of human genome DNA orvarious human tissue-derived cDNAs by random sequencing and rapidprogress in gene analysis technology have been accelerating theinvestigation of human genome. With such advance, it has been clarifiedthat there are many genes supposed to encode proteins with unknownfunctions. G protein-coupled receptors not only have seven transmembranedomains but many common sequences are present in their nucleic acids oramino acids. Thus, these receptors can be precisely identified to be Gprotein-coupled receptors in such proteins. On the other hand, these Gprotein-coupled receptor genes are obtained also by polymerase chainreaction (hereinafter abbreviated as PCR) utilizing such a structuralsimilarity. In these G protein-coupled receptors thus obtained so far,ligands to some receptors that are subtypes having high homology instructure to known receptors may be readily predictable but in mostcases, their endogenous ligands are unpredictable so that ligandscorresponding to these receptors are not found. For this reason, thesereceptors are termed orphan receptors. It is likely that unidentifiedendogenous ligands to such orphan receptors would participate inbiological phenomena poorly analyzed because the ligands were unknown.When such ligands are associated with important physiological effects orpathologic conditions, it is expected that development of these receptoragonists or antagonists will result in breakthrough new drugs (Stadel,J. et al., TiPS, 18, 430-437, 1997; Marchese, A. et al., TiPS, 20,370-375, 1999; Civelli, O. et al., Brain Res., 848, 63-65, 1999, Howard,A. D. et al, TiPS, 22, 132-140, 2001).

Recently, some groups attempted to investigate ligands to these orphanreceptors and reported isolation/structural determination of ligands,which are novel physiologically active peptides. Independently,Reinsheid et al. and Meunier et al. introduced a cDNA coding for orphanG protein-coupled receptor LC132 or ORL1 into animal cells to express areceptor, isolated a novel peptide from porcine brain or rat brainextract, which was named orphanin FQ or nociceptin, with reference toits response and determined its sequence (Reinsheid, R. K. et al.,Science, 270, 792-794, 1995; Meunier, J.-C. et al., Nature, 377,532-535, 1995). This peptide was reported to be associated with pain.Further research on the receptor in knockout mice reveals that thepeptide takes part in memory (Manabe, T. et al., Nature, 394, 577-581,1998).

Subsequently, novel peptides such as PrRP (prolactin releasing peptide),orexin, apelin, ghrelin and GALP (galanin-like peptide), etc. wereisolated as ligands to orphan G protein-coupled receptors (Hinuma, S. etal., Nature, 393, 272-276, 1998; Sakurai, T. et al., Cell, 92, 573-585,1998; Tatemoto, K. et al., Biohem. Biophys. Res. Commun., 251, 471476,1998; Kojima, M. et al., Nature, 402, 656-660, 1999; Ohtaki, T. et al.,J. Biol. Chem., 274, 37041-37045, 1999). On the other hand, somereceptors to physiologically active peptides, which were hithertounknown, were clarified. It was revealed that a receptor to motilinassociated with contraction of intestinal tracts was GPR38 (Feighner, S.D. et al., Science, 284, 2184-2188, 1999). Furthermore, SLC-1 wasidentified to be a receptor to MCH (Chambers, J. et al., Nature, 400,261-265, 1999; Saito, Y. et al., Nature, 400, 265-269, 1999; Shimomura,Y. et al., Biochem. Biophys. Res. Commun. 261, 622-626, 1999; Lembo, P.M. C. et al., Nature Cell Biol., 1, 267-271, 1999; Bachner, D. et al.,FEBS Lett., 457, 522-524, 1999). Also, GPR14 (SENR) was reported to be areceptor to urotensin II (Ames, R. S. et al., Nature, 401, 282-286,1999; Mori, M. et al., Biochem. Biophys. Res. Commun., 265, 123-129,1999; Nothacker, H.-P. et al., Nature Cell Biol., 1, 383-385, 1999, Liu,Q. et al., Biochem. Biophys. Res. Commun., 266, 174-178, 1999). Besides,receptors to neuromedin U and neuropetide FF, which are neuropeptides,have recently been clarified and furthermore, low molecularphysiologically active lipids or nucleic acid derivatives such ascysteinyl leukotrienes, sphingosine-1-phosphate, lysophosphatidic acid,sphingosylphosphorylcholine, UDP-glucose, etc., have been identified tobe ligands to orphan receptors (Howard, A. D. et al., TiPS, 22, 132-140,2001). It was shown that MCH took part in obesity since its knockoutmice showed the reduced body weight and lean phenotype (Shimada, M. etal., Nature, 396, 670-674, 1998), and because its receptor was revealed,it became possible to explore a receptor antagonist likely to be anantiobesity agent. It is also reported that urotensin II shows a potentaction on the cardiocirculatory system, since it induces heart ischemiaby intravenous injection to monkey (Ames, R. S. et al., Nature, 401,282-286, 1999).

As described above, orphan receptors and ligands thereto often take partin a new physiological activity, and it is expected that theirclarification will lead to development of new drugs. However, it isknown that research on ligands to orphan receptors is accompanied bymany difficulties. For example, it is generally unknown what secondarysignal transduction system will take place after orphan receptorsexpressed on cells responded to ligands, and various response systemshould be examined. Moreover, tissues where ligands are present are notreadily predictable so that various tissue extracts should be prepared.Furthermore, since an amount of ligand required to stimulate itsreceptor is sufficient even in an extremely low concentration when theligand is a peptide, the amount of such a ligand present in vivo is atrace amount in many cases. In addition, a peptide is digested bypeptidase to lose its activity, or undergoes non-specific adsorption sothat its recovery becomes poor during purification. Normally, it is thusextremely difficult to extract such a ligand from the living body andisolate an amount of the ligand necessary for determination of itsstructure. The presence of many orphan receptors was unraveled, but onlya very small part of ligands to these receptors were discovered so fardue to the foregoing problems.

GPR7 is one of the reported orphan G protein-coupled receptors (SEQ IDNO:49, O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995). GPR7 has a lowhomology to somatostatin receptor (SSTR3) and opioid receptors (δ, κ andμ). Also, GPR7 is found to have a homology of about 64% to GPR8 (SEQ IDNO:66, O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995) on an amino acidlevel. It is reported by O'Dowd, B. F. et al. that [³H] bremazocinebinds to the membrane fraction of GPR7 and this binding is inhibited byβ-funaltrexamine, [D-Pro4]morphiceptin or β-endorphin, which is aμ-opioid receptor selective ligand, U50 or 488, which is a κ-opioidreceptor selective ligand, or naltrindole, which is a δ-opioid receptorselective ligand.

The present invention provides a novel peptide capable of binding toGPR7, etc., its DNA, a method of screening a drug using the peptide andGPR7, etc.

DISCLOSURE OF THE INVENTION

The present inventors made extensive studies to solve the foregoingproblems. As a result, the inventors succeeded in acquiring DNAsencoding novel peptides (GPR7 ligands) capable of binding to GPR7, fromhuman whole brain, mouse whole brain and rat whole brain, and found thatthe GPR7 ligands exhibit an appetite (eating) stimulating activity. Inaddition, the inventors succeeded in acquiring DNAs encoding GPR7 andGPR8, respectively, from bovine hypothalamus. As a result of furtherstudies based on these findings, the inventors have come to accomplishthe present invention.

That is, the present invention provides the following features:

(1) A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:1 whereinthe N-terminal amino acid residue may optionally be brominated, or itsamide or ester, or a salt thereof;

(2) The peptide or its amide or ester, or a salt thereof, according to(1), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:1;

(3) The peptide or its amide or ester, or a salt thereof, according to(1), which has the amino acid sequence represented by SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3 or SEQ ID NO:66;

(4) The peptide or its amide or ester, or a salt thereof, according to(1), wherein the N-terminal tryptophan residue is 6-brominated and whichhas the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3 or SEQ ID NO:66;

(5) A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:4 whereinthe N-terminal amino acid residue may optionally be brominated, or itsamide or ester, or a salt thereof;

(6) The peptide or its amide or ester, or a salt thereof, according to(5), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:4;

(7) The peptide or its amide or ester, or a salt thereof, according to(5), which has the amino acid sequence represented by SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 or SEQ ID NO:67;

(8) The peptide or its amide or ester, or a salt thereof, according to(5), wherein the N-terminal tryptophan residue is 6-brominated and whichhas the amino acid sequence represented by SEQ ID NO:67;

(9) The peptide or its amide or ester, or a salt thereof, according to(5), wherein the N-terminal tryptophan residue is 6-brominated and whichhas the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 or SEQ ID NO:67;

(10) A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:7 whereinthe N-terminal amino acid residue may optionally be brominated, or itsamide or ester, or a salt thereof;

(11) The peptide or its amide or ester, or a salt thereof, according to(10), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:7;

(12) The peptide or its amide or ester, or a salt thereof, according to(10), which has the amino acid sequence represented by SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:68 or SEQ ID NO:69;

(13) A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:13 whereinthe N-terminal amino acid residue may optionally be brominated, or itsamide or ester, or a salt thereof;

(14) The peptide or its amide or ester, or a salt thereof, according to(13), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:13;

(15) The peptide or its amide or ester, or a salt thereof, according to(13), which has the amino acid sequence represented by SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15 or SEQ ID NO:70;

(16) A peptide containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:16 whereinthe N-terminal amino acid residue may optionally be brominated, or itsamide or ester, or a salt thereof;

(17) The peptide or its amide or ester, or a salt thereof, according to(16), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:16;

(18) The peptide or its amide or ester, or a salt thereof, according to(17), which has the amino acid sequence represented by SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18 or SEQ ID NO:71;

(19) The peptide or its amide or ester, or a salt thereof, according to(1) through (18), which is capable of binding to a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:49 or SEQ ID NO:86;

(20) The peptide or its amide or ester, or a salt thereof, according to(1) through (18), which is capable of binding to a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:59;

(21) The peptide or its amide or ester, or a salt thereof, according to(1) through (18), which is capable of binding to a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:84 or SEQ ID NO:88;

(22) A partial peptide of the peptide according to any one of (1)through (21), or its amide or ester, or a salt thereof;

(23) A precursor peptide of the peptide according to any one of (1)through (21), or its amide or ester, or a salt thereof;

(24) The precursor peptide or its amide or ester, or a salt thereof,according to (23), which contains the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:19;

(25) The peptide or its amide or ester, or a salt thereof, according to(24), which has the amino acid sequence represented by SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21 or SEQ ID NO:72;

(26) The peptide or its amide or ester, or a salt thereof, according to(23), which contains the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:22;

(27) The peptide or its amide or ester, or a salt thereof, according to(26), which has the amino acid sequence represented by SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24 or SEQ ID NO:73;

(28) A polynucleotide containing a polynucleotide encoding the peptideaccording to any one of (1) through (21);

(29) The polynucleotide according to (28), which has the base sequencerepresented by SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79;

(30) A polynucleotide containing a polynucleotide encoding the partialpeptide according to (22);

(31) A polynucleotide containing a polynucleotide encoding the precursorpeptide according to (23);

(32) The polynucleotide according to (31), which has the base sequencerepresented by SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:80 or SEQ ID NO:81;

(33) The polynucleotide according to (28) to (32), which is a DNA;

(34) A recombinant vector containing the polynucleotide according to anyone of (28) to (33);

(35) A transformant transformed with the recombinant vector according to(34);

(36) A method of manufacturing the peptide, its partial peptide or itsprecursor peptide, or a salt thereof, according to any one of (1) to(21), which comprises culturing the transformant according to (35) andproducing the peptide, partial peptide or precursor peptide according toany one of (1) to (21);

(37) An antibody to the peptide, its partial peptide or its precursorpeptide, or its amide or ester, or a salt thereof, according to any oneof (1) to (21);

(38) The antibody according to (37), which is a neutralizing antibody toinactivate the activity of the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21);

(39) A pharmaceutical comprising the antibody according to (37);

(40) The pharmaceutical according to (39), which is apreventive/therapeutic agent for obesity or hyperphagia;

(41) A diagnostic product comprising the antibody according to (37);

(42) The diagnostic product according to (41), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(43) A pharmaceutical comprising the peptide, its partial peptide, orits amide or ester, or a salt thereof, according to any one of (1) to(21);

(44) The pharmaceutical according to (43), which is apreventive/therapeutic agent for anorexia or eating stimulant;

(45) A pharmaceutical comprising the polynucleotide according to (28);

(46) The pharmaceutical according to (45), which is apreventive/therapeutic agent for anorexia or eating stimulant;

(47) A diagnostic product comprising the polynucleotide according to(28);

(48) The diagnostic product according to (47), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(49) A polynucleotide containing a complementary base sequence to thepolynucleotide according to (28), or a part thereof;

(50) A pharmaceutical comprising the polynucleotide according to (49);

(51) The pharmaceutical according to (50), which is apreventive/therapeutic agent for obesity or hyperphagia;

(52) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49;

(53) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59;

(54) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84;

(55) A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:49;

(56) A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:59;

(57) A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:84;

(58) A compound or its salt that alters the binding property between thepeptide, its partial peptide, or its amide or ester, or a salt thereof,according to any one of (1) to (21) and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:49, which is obtainable by using thescreening method according to (52) or the screening kit according to(55);

(59) A compound or its salt that alters the binding property between thepeptide, its partial peptide, or its amide or ester, or a salt thereof,according to any one of (1) to (21) and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:59, which is obtainable by using thescreening method according to (53) or the screening kit according to(56);

(60) A compound or its salt that alters the binding property between thepeptide, its partial peptide, or its amide or ester, or a salt thereof,according to any one of (1) to (21) and a protein or its salt containingthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:84, which is obtainable by using thescreening method according to (54) or the screening kit according to(57);

(61) The compound or its salt according to (58) to (60), which is anagonist;

(62) The compound or its salt according to (58) to (60), which is anantagonist;

(63) A pharmaceutical comprising the compound or its salt according toany one of (58) to (60);

(64) A preventive/therapeutic agent for anorexia or eating stimulantcomprising the agonist according to (61);

(65) A preventive/therapeutic agent for obesity or hyperphagiacomprising; the antagonist according to (62);

(66) An antiobesity agent obtainable by using the screening methodaccording to (52) or the screening kit according to (55);

(67) An antiobesity agent obtainable by using the screening methodaccording to (53) or the screening kit according to (56);

(68) An antiobesity agent obtainable by using the screening methodaccording to (54) or the screening kit according to (57);

(69) A method of screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to any one of (1) to (21), which comprises using a DNAencoding the peptide, its partial peptide or its precursor peptideaccording to any one of (1) to (21);

(70) A kit for screening a compound or its salt that alters theexpression level of the peptide, its partial peptide or its precursorpeptide according to any one of (1) to (21), comprising a DNA encodingthe peptide, its partial peptide or its precursor peptide according toany one of (1) to (21);

(71) A compound or its salt that alters the expression level of thepeptide, its partial peptide or its precursor peptide according to anyone of (1) to (21), which is obtainable by using the screening methodaccording to (69) or the screening kit according to (70);

(72) The compound or its salt according to (71), which is a compound orits salt that increases the expression level;

(73) The compound or its salt according to (71), which is a compound orits salt that decreases the expression level;

(74) A pharmaceutical comprising the compound or its salt according to(71);

(75) A preventive/therapeutic agent for anorexia or eating stimulantcomprising the compound or its salt according to (72);

(76) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (73);

(77) A method for preventing/treating anorexia, which comprisesadministering to a mammal an effective amount of the peptide, itspartial peptide, or its amide or ester, or a salt thereof, according toany one of (1) to (21), the polynucleotide according to (28), theagonist according to (61), or the compound or its salt according to(72);

(78) A method for stimulating appetite, which comprises administering toa mammal an effective amount of the peptide, its partial peptide, or itsamide or ester, or a salt thereof, according to any one of (1) to (21),the polynucleotide according to (28), the agonist according to (61), orthe compound or its salt according to (72);

(79) A method for preventing/treating obesity or hyperphagia, whichcomprises administering to a mammal an effective amount of the antibodyaccording to (37), the polynucleotide according to (49), the antagonistaccording to (62), or the compound or its salt according to (73);

(80) A protein or its salt containing the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:86;

(81) The protein or its salt according to (80), containing the aminoacid sequence represented by SEQ ID NO:86;

(82) A partial peptide or its salt of the protein according to (80);

(83) A polynucleotide containing a polynucleotide encoding the proteinaccording to (80), or a partial peptide thereof;

(84) The polynucleotide according to (83), which is a DNA;

(85) The polynucleotide according to (84), which contains the basesequence represented by SEQ ID NO:87;

(86) A recombinant vector containing the polynucleotide according to(83);

(87) A transformant transformed by the recombinant vector according to(86);

(88) A method of manufacturing the protein according to (80), itspartial peptide or a salt thereof, which comprises culturing thetransformant according to (87) and producing the protein according to(80), its partial peptide or a salt thereof;

(89) A pharmaceutical comprising the protein according to (80) or thepartial peptide according to (82), or a salt thereof;

(90) A pharmaceutical comprising the polynucleotide according to (83);

(91) The pharmaceutical according to (90), which is apreventive/therapeutic agent for anorexia or eating stimulant;

(92) A diagnostic product comprising the polynucleotide according to(83);

(93) The diagnostic product according to (92), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(94) An antibody to the protein according to (80) or the partial peptideaccording to (82), or a salt thereof;

(95) The antibody according to (94), which is a neutralizing antibody toinactivate signal transduction of the protein according to (80);

(96) A pharmaceutical comprising the antibody according to (94);

(97) The pharmaceutical according to (96), which is apreventive/therapeutic agent for obesity or hyperphagia;

(98) A diagnostic product comprising the antibody according to (94);

(99) The diagnostic product according to (99), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(100) A polynucleotide containing a complementary base sequence to thepolynucleotide according to (83), or a part thereof;

(101) A pharmaceutical comprising the polynucleotide according to (100);

(102) The pharmaceutical according to (101), which is apreventive/therapeutic agent for obesity or hyperphagia;

(103) A protein or its salt containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88;

(104) The protein or its salt according to (103), which contains theamino acid sequence represented by SEQ ID NO:88;

(105) A partial peptide of the protein according to (103), or a saltthereof;

(106) A polynucleotide containing a polynucleotide encoding the proteinaccording to (103) or a partial peptide thereof;

(107) The polynucleotide according to (106), which is a DNA;

(108) The polynucleotide according to (107), which contains the basesequence represented by SEQ ID NO:89;

(109) A recombinant vector containing the polynucleotide according to(108);

(110) A transformant transformed with the recombinant vector accordingto (109);

(111) A method of manufacturing the protein according to (103), itspartial peptide, or a salt thereof, which comprises culturing thetransformant of (110) and producing the protein according to (103) orits partial peptide;

(112) A pharmaceutical comprising the protein according to (103) or thepartial peptide according to (105), or a salt thereof;

(113) A pharmaceutical comprising the polynucleotide according to (106);

(114) The pharmaceutical according to (113), which is apreventive/therapeutic agent for anorexia or eating stimulant;

(115) A diagnostic product comprising the polynucleotide according to(106);

(116) The diagnostic product according to (115), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(117) An antibody to the protein according to (103) or the partialpeptide according to (105), or a salt thereof;

(118) The antibody according to (117), which is a neutralizing antibodyto inactivate signal transduction of the protein according to (103);

(119) A pharmaceutical comprising the antibody according to (117);

(120) The pharmaceutical according to (119), which is apreventive/therapeutic agent for obesity or hyperphagia;

(121) A diagnostic product comprising the antibody according to (117);

(122) The diagnostic product according to (121), which is a diagnosticproduct for anorexia, obesity or hyperphagia;

(123) A polynucleotide containing a complementary base sequence to thepolynucleotide according to (106), or a part thereof;

(124) A pharmaceutical comprising the polynucleotide according to (123);

(125) The pharmaceutical according to (124), which is apreventive/therapeutic agent for obesity or hyperphagia;

(126) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:86,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:86;

(127) A method of screening a compound or its salt that alters thebinding property between the peptide, its partial peptide, or its amideor ester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88,which comprises using the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88;

(128) A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:86,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:86;

(129) A kit for screening a compound or its salt that alters the bindingproperty between the peptide, its partial peptide, or its amide orester, or a salt thereof, according to any one of (1) to (21) and aprotein or its salt containing the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:88,comprising the peptide, its partial peptide, or its amide or ester, or asalt thereof, according to any one of (1) to (21) and a protein or itssalt containing the same or substantially the same amino acid sequenceas the amino acid sequence represented by SEQ ID NO:88;

(130) A compound or its salt that alters the binding property betweenthe peptide, its partial peptide, or its amide or ester, or a saltthereof, according to any one of (1) to (21) and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:86, which is obtainable byusing the screening method according to (126) or the screening kitaccording to (128);

(131) A compound or its salt that alters the binding property betweenthe peptide, its partial peptide, or its amide or ester, or a saltthereof, according to any one of (1) to (21) and a protein or its saltcontaining the same or substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:88, which is obtainable byusing the screening method according to (127) or the screening kitaccording to (129);

(132) The compound or its salt according to (130) or (131), which is anagonist;

(133) The compound or its salt according to (130) or (131), which is anantagonist;

(134) A pharmaceutical comprising the compound or its salt according to(130) or (131);

(135) A preventive/therapeutic agent for anorexia or eating stimulant,comprising the agonist according to (132);

(136) A preventive/therapeutic agent for obesity or hyperphagia,comprising the antagonist according to (133);

(137) A method of screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, which comprises using a DNA encoding a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:86;

(138) A kit for screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:86, comprising a DNA encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:86;

(139) A compound or its salt that alters the expression level of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:86, whichis obtainable by using the screening method according to (137) or thescreening kit according to (138);

(140) The compound or its salt according to (139), which is a compoundor its salt that increases the expression level;

(141) The compound or its salt according to (139), which is a compoundor its salt that decreases the expression level;

(142) A pharmaceutical comprising the compound or its salt according to(139);

(143) A preventive/therapeutic agent for anorexia or eating stimulantcomprising the compound or its salt according to (140);

(144) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (141);

(145) A method of screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88, which comprises using a DNA encoding a protein containing thesame or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:88;

(146) A kit for screening a compound or its salt that alters theexpression level of a protein containing the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88, comprising a DNA encoding a protein containing the same orsubstantially the same amino acid sequence as the amino acid sequencerepresented by SEQ ID NO:88;

(147) A compound or its salt that alters the expression level of aprotein containing the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:88, whichis obtainable by using the screening method according to (145) or thescreening kit according to (146);

(148) The compound or its salt according to (147), which is a compoundor its salt that increases the expression level;

(149) The compound or its salt according to (147), which is a compoundor its salt that decreases the expression level;

(150) A pharmaceutical comprising the compound or its salt according to(147);

(151) A preventive/therapeutic agent for anorexia or eating stimulantcomprising the compound or its salt according to (148);

(152) A preventive/therapeutic agent for obesity or hyperphagiacomprising the compound or its salt according to (149);

(153) A method for preventing/treating anorexia, which comprisesadministering to a mammal an effective amount of the protein accordingto (80), its partial peptide or a salt thereof, the polynucleotideaccording to (83), the protein according to (103), its partial peptideor a salt thereof, the polynucleotide according to (106), the agonistaccording to (132), the compound or its salt according to (140), or thecompound or its salt according to (148);

(154) A method for stimulating appetite, which comprises administeringto a mammal an effective amount of the protein according to (80), itspartial peptide or a salt thereof, the polynucleotide according to (83),the protein according to (103), its partial peptide or a salt thereof,the polynucleotide according to (106), the agonist according to (132),the compound or its salt according to (140), or the compound or its saltaccording to (148);

(155) A method for preventing/treating obesity or hyperphagia, whichcomprises administering to a mammal an effective amount of the antibodyaccording to (94), the polynucleotide according to (100), the antibodyaccording to (117), the polynucleotide according to (123), theantagonist according to (133), the compound or its salt according to(141), or the compound or its salt according to (149);

(156) A non-human mammal bearing the DNA according to (28), which isexogenous, or its variant DNA;

(157) The mammal according to (156), wherein the non-human mammal is arodent;

(158) A recombinant vector bearing the exogenous DNA or its variant DNAaccording to (28) and capable of expressing in a mammal;

(159) A non-human embryonic stem cell, wherein the DNA according to (28)is inactivated;

(160) The embryonic stem cell according to (159), wherein the DNA isinactivated by introducing a reporter gene;

(161) The embryonic stem cell according to (159), wherein the non-humanmammal is a rodent;

(162) A non-human mammal deficient in expressing the DNA according to(28), wherein the DNA is inactivated;

(163) A non-human mammal according to (162), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (28);

(164) The non-human mammal according to (162), which is a rodent;

(165) A method of screening a compound or its salt that promotes orinhibits the activity of a promoter for the DNA according to (28), whichcomprises administering a test compound to the mammal according to (163)and detecting expression of the reporter gene;

(166) A non-human mammal bearing the DNA according to (83), which isexogenous, or its variant DNA;

(167) The mammal according to (166), wherein the non-human mammal is arodent;

(168) A recombinant vector bearing the exogenous DNA or its variant DNAaccording to (83) and capable of expressing in a mammal;

(169) A non-human embryonic stem cell, wherein the DNA according to (83)is inactivated;

(170) The embryonic stem cell according to (169), wherein the DNA isinactivated by introducing a reporter gene;

(171) The embryonic stem cell according to (169), wherein the non-humanmammal is a rodent;

(172) A non-human mammal deficient in expressing the DNA according to(83), wherein the DNA is inactivated;

(173) A non-human mammal according to (172), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (83);

(174) The non-human mammal according to (172), which is a rodent;

(175) A method of screening a compound or its salt that promotes orinhibits the activity of a promoter for the DNA according to (83), whichcomprises administering a test compound to the mammal according to (173)and detecting expression of the reporter gene;

(176) A non-human mammal bearing the DNA according to (106), which isexogenous, or its variant DNA;

(177) The mammal according to (176), wherein the non-human mammal is arodent;

(178) A recombinant vector bearing the exogenous DNA according to (106)or its variant DNA and capable of expressing in a mammal;

(179) A non-human embryonic stem cell, wherein the DNA according to(106) is inactivated;

(180) The embryonic stem cell according to (179), wherein the DNA isinactivated by introducing a reporter gene;

(181) The embryonic stem cell according to (179), wherein the non-humanmammal is a rodent;

(182) A non-human mammal deficient in expressing the DNA according to(106), wherein the DNA is inactivated;

(183) The non-human mammal according to (182), wherein the DNA isinactivated by inserting a reporter gene therein and the reporter geneis capable of expressing under control of a promoter for the DNAaccording to (106);

(184) The non-human mammal according to (182), which is a rodent;

(185) A method of screening a compound or its salt that promotes orinhibits the activity of a promoter for the DNA according to (106),which comprises administering a test compound to the mammal according to(183) and detecting expression of the reporter gene;

(186) Use of the peptide, its partial peptide, its amide or ester, or asalt thereof, according to any one of (1) through (21), thepolynucleotide according to (28), the agonist according to (61), or thecompound or its salt according to (72), for manufacturing apreventive/therapeutic agent for anorexia;

(187) Use of the peptide, its partial peptide, its amide or ester, or asalt thereof, according to any one of (1) through (21), thepolynucleotide according to (28), the agonist according to (61), or thecompound or its salt according to (72), for manufacturing an eatingstimulant;

(188) Use of the antibody according to (37), the polynucleotideaccording to (49), the antagonist according to (62), or the compound orits salt according to (73), for manufacturing a preventive/therapeuticagent for obesity or hyperphagia;

(189) Use of the protein according to (80) or its partial peptide or asalt thereof, the polynucleotide according to (83), the proteinaccording to (103) or its partial peptide or a salt thereof, thepolynucleotide according to (106), the agonist according to (132), thecompound or its salt according to (140) or the compound or its saltaccording to (148), for manufacturing a preventive/therapeutic agent foranorexia;

(190) Use of the protein according to (80) or its partial peptide or asalt thereof, the polynucleotide according to (83), the proteinaccording to (103) or its partial peptide or a salt thereof, thepolynucleotide according to (106), the agonist according to (132), thecompound or its salt according to (140) or the compound or its saltaccording to (148), for manufacturing an eating stimulant; and,

(191) Use of the antibody according to (94), the polynucleotideaccording to (100), the antibody according to (117), the polynucleotideaccording to (123), the antagonist according to (133), the compound orits salt according to (141), or the compound or its salt according to(149), for manufacturing a preventive/therapeutic agent for obesity orhyperphagia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence (SEQ ID NO: 101) of human GPR7 ligandprecursor H.

FIG. 2 shows the amino acid sequence (SEQ ID NO: 22) of human GPR7ligand precursor H.

FIG. 3 shows the DNA sequence (SEQ ID NO: 102) of mouse GPR7 ligandprecursor H.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 23) of mouse GPR7ligand precursor H.

FIG. 5 shows the DNA sequence (SEQ ID NO: 103) of rat GPR7 ligandprecursor H.

FIG. 6 shows the amino acid sequence (SEQ ID NO: 24) of rat GPR7 ligandprecursor H.

FIG. 7 shows a comparison between human (SEQ ID NO: 22), rat (SEQ ID NO:24) and mouse (SEQ ID NO: 23) ligand precursors H, wherein the matchedamino acids are indicated in a box, and the arrow indicates thepredicted cleavage site of a secretion signal.

FIG. 8 shows the results of suppression detected on a luciferaseactivity by ligand stimulation, when the culture supernatants of ligandexpression vector pAK-S64 and empty expression vector(pAKKO-111H)-expressed CHO cells were added in the presence of forskolin(FSK) to a medium of CHO cells wherein GPR7 cDNA-inserted plasmid wastransiently expressed.

FIG. 9 shows the results of suppression detected on a luciferaseactivity, when he culture supernatant of CHO cells wherein S64 wastransiently expressed was added in the presence of forskolin (FSK) to amedium of CHO cells wherein TGR26 was transiently expressed.

FIG. 10 shows the results of GPR7-expressed CHO cell-specific cAMPproduction level suppression caused by the supernatant of cells whereinS64 was transiently expressed.

FIG. 11 shows the results of cAMP production level suppression of mockCHO caused by the supernatant of cells wherein S64 was transientlyexpressed.

FIG. 12 shows the tissue distribution of GPR7 ligand mRNA in rat and theresults of its expression level as determined by RT-PCR.

FIG. 13 shows a chromatogram of endogenous GPR7 ligand finally purifiedfrom bovine hypothalamus, which indicates the chromatographic pattern onμRPC C2/C18 SC 2.1/10 at the final step of purification and the cAMPproduction level assayed using the cAMP-screen system (ABI) afterreacting each fraction with human GPR7-expressed CHO cells. Thechromatographic pattern shows the absorbance at 215 nm and the elutionconcentration of acetonitrile.

FIG. 14 shows the results of N-terminal sequencing of the endogenousGPR7 ligand purified from bovine hypothalamus.

FIG. 15 shows an ESIMS spectrum (upper column) and a MS/MS spectrum(lower column) of the endogenous GPR7 ligand purified from bovinehypothalamus.

FIG. 16 shows a zoom scanning spectrum of trivalent molecular ions.

FIG. 17 shows the results of standard analysis performed by mixingPTH-5-bromotryptophan (5BrW) and PTH-6-bromotryptophan (6BrW) with 20amino acid PTH standards (peaks shown by asterisk*), whereby it wasconfirmed that the peaks of standard 5BrW overlapped with those of 5BrWin an unknown sample.

FIG. 18 shows the results of standard analysis performed by mixingPTH-5-bromotryptophan (5BrW) and PTH-6-bromotryptophan (6BrW) with 20amino acid PTH standards (peaks shown by asterisk*), whereby it wasconfirmed that the peaks of standard 6BrW overlapped with those of 6BrWin an unknown sample.

FIG. 19 shows the results of N-terminal sequencing of GPR7L purifiedfrom bovine hypothalamus, wherein the amino acids by standard analysisand at cycle 1 on the chromatogram up to cycle 2 coincided with thepeaks of 6-bromotryptophan.

FIG. 20 shows a DNA sequence (SEQ ID NO: 104) of bovine GPR7 ligandprecursor H.

FIG. 21 shows an amino acid sequence (SEQ ID NO: 73) of bovine GPR7ligand precursor H.

FIG. 22 shows change in feed uptake with passage of time every 2 hoursafter non-brominated GPR7L or distilled water was administered to ratinto the lateral ventricle, wherein Vehicle and bGPR7L (Br—) indicatedistilled water and non-brominated bovine GPR7 ligand, respectively.

FIG. 23 shows a FMS spectrum of endogenous GPR7 ligand.

FIG. 24 shows the results of the tissue distribution and expressionlevel of GPR7 ligand mRNA in human as determined by RT-PCR.

FIG. 25 shows the results of the tissue distribution and expressionlevel of rat GPR7 (rat TGR26) mRNA as determined by RT-PCR.

FIG. 26 shows a cDNA sequence (SEQ ID NO: 105) of bovine GPR7.

FIG. 27 shows an amino acid sequence (SEQ ID NO: 86) of bovine GPR7.

FIG. 28 shows a cDNA sequence (SEQ ID NO: 106) of bovine GPR8.

FIG. 29 shows an amino acid sequence (SEQ ID NO: 88) of bovine GPR8.

FIG. 30 shows the results of final purification of human GPR7 ligandfrom the culture supernatant of human GPR7 ligand-expressed CHO cells,wherein chromatographic pattern of μRPC C2C18 SC2.1/10 at the finalpurification step and the specific intracellular cAMP productionsuppression activity obtained by recating each fraction with humanGPR7-expressed CHO cells are shown. On the chromatogram, absorbance at215 nm and the elution concentration of acetonitrile are shown.

FIG. 31 shows the results of N-terminal sequencing (residues 1-20 of SEQID NO: 1) of GPR7 ligand purified from the culture supernatant of humanGPR7 ligand-expressed CHO cells.

FIG. 32 shows an ESI-MS spectrum of GPR7 ligand purified from theculture supernatant of human GPR7 ligand-expressed CHO cells.

FIG. 33 shows the results of Scatchard analysis using human GPR7ligand-expressed CHO cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The peptide of the present invention having the same or substantiallythe same amino acid sequence as the amino acid sequence represented bySEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:16(hereinafter sometimes merely referred to as the peptide of the presentinvention) may be any peptide derived from any cells of human or otherwarm-blooded animals, e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc. (e.g., retina cells, liver cells,splenocytes, nerve cells, glial cells, β cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells,immune cells (e.g., macrophages, T cells, B cells, natural killer cells,mast cells, neutrophils, basophils, eosinophils, monocytes),megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts,osteoclasts, mammary gland cells, hepatocytes or interstitial cells, thecorresponding precursor cells, stem cells, cancer cells, etc., of thesecells), or any tissues where such cells are present, e.g., brain or anyregion of the brain (e.g., retina, olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; or hemocyte typecells or their cultured cells (e.g., MEL, M1, CTLL-2, HT-2, WEHI-3,HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1,Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL,JK-1, CMK, KO-812, MEG-01, etc.). The peptide may also be a syntheticpeptide.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:13 or SEQ ID NO:16 includes an amino acid sequence having atleast about 70% homology, preferably at least about 80% homology, morepreferably at least about 90% homology, and most preferably at leastabout 95% homology, to the amino acid sequence represented by SEQ IDNO:1.

As the peptide having substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:1, preferred is a peptidehaving substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:1 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:1, etc.

As the peptide having substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:4, preferred is a peptidehaving substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:4 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:4, etc.

As the peptide having substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:7, preferred is a peptidehaving substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:7 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:7, etc.

As the peptide having substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:13, preferred is a peptidehaving substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:13 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:13, etc.

As the peptide having substantially the same amino acid sequence as theamino acid sequence represented by SEQ ID NO:16, preferred is a peptidehaving substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:16 and having the activitysubstantially equivalent to that of the amino acid sequence representedby SEQ ID NO:16, etc.

Specifically, the substantially equivalent activity includes activitiesthat the peptide of the present invention possesses (for example,preventive/therapeutic activities for diseases described below, GPR7binding activities, cell stimulating activities on the GPR7-expressedcells (e.g., activities that promote arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, GTPγS bindingactivities, etc.) and the like.

The term substantially equivalent is used to mean that these activitiesare equivalent in nature (e.g., biochemically or pharmacologically).

Specific examples of the amino acid sequence, which is substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:1, include:

(i) the amino acid sequence represented by SEQ ID NO:1;

(ii) the amino acid sequence represented by SEQ ID NO:2;

(iii) the amino acid sequence represented by SEQ ID NO:3;

(iv) the amino acid sequence represented by SEQ ID NO:66;

(v) the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:66, to which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:66, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are replaced byother amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

Specific examples of the amino acid sequence, which is substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:4, include:

(i) the amino acid sequence represented by SEQ ID NO:4;

(ii) the amino acid sequence represented by SEQ ID NO:5;

(iii) the amino acid sequence represented by SEQ ID NO:6;

(iv) the amino acid sequence represented by SEQ ID NO:67;

(v) the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6 or SEQ ID NO:67, to which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6 or SEQ ID NO:67, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are replaced byother amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

Specific examples of the amino acid sequence, which is substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:7, include:

(i) the amino acid sequence represented by SEQ ID NO:7;

(ii) the amino acid sequence represented by SEQ ID NO:8;

(iii) the amino acid sequence represented by SEQ ID NO:9;

(iv) the amino acid sequence represented by SEQ ID NO:10;

(v) the amino acid sequence represented by SEQ ID NO:11;

(vi) the amino acid sequence represented by SEQ ID NO:12;

(vii) the amino acid sequence represented by SEQ ID NO:68;

(viii) the amino acid sequence represented by SEQ ID NO:69;

(ix) the amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:68 orSEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, more preferably 1 to2, and most preferably 1) amino acids are deleted;

(x) the amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:68 or SEQID NO:69, to which 1 to 5 (preferably 1 to 3, more preferably 1 to 2,and most preferably 1) amino acids are added;

(xi) the amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:68 orSEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, more preferably 1 to2, and most preferably 1) amino acids are inserted;

(xii) the amino acid sequence represented by SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:68 orSEQ ID NO:69, in which 1 to 5 (preferably 1 to 3, more preferably 1 to2, and most preferably 1) amino acids are replaced by other amino acids;

(xiii) amino acid sequences in combination of (ix) to (xii) above; etc.

Specific examples of the amino acid sequence, which is substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:13, include:

(i) the amino acid sequence represented by SEQ ID NO:13;

(ii) the amino acid sequence represented by SEQ ID NO:14;

(iii) the amino acid sequence represented by SEQ ID NO:15;

(iv) the amino acid sequence represented by SEQ ID NO:70;

(v) the amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 or SEQ ID NO:70, to which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 0.1) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15 or SEQ ID NO:70, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are replacedby other amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

Specific examples of the amino acid sequence, which is substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:16, include:

(i) the amino acid sequence represented by SEQ ID NO:16;

(ii) the amino acid sequence represented by SEQ ID NO:17;

(iii) the amino acid sequence represented by SEQ ID NO:18;

(iv) the amino acid sequence represented by SEQ ID NO:71;

(v) the amino acid sequence represented by SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18 or SEQ ID NO:71, to which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to 3, morepreferably 1 to 2, and most preferably 1) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 or SEQ ID NO:71, in which 1 to 5 (preferably 1 to 3,more preferably 1 to 2, and most preferably 1) amino acids are replacedby other amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

Specific examples of the peptide of the present invention are, forexample:

[Peptide A]

human peptide having the amino acid sequence represented by SEQ ID NO:1;

mouse peptide having the amino acid sequence represented by. SEQ IDNO:2;

rat peptide having the amino acid sequence represented by SEQ ID NO:3;

bovine peptide having the amino acid sequence represented by SEQ IDNO:66;

[Peptide B]

human peptide having the amino acid sequence represented by SEQ ID NO:4;

mouse peptide having the amino acid sequence represented by SEQ ID NO:5;

rat peptide having the amino acid sequence represented by SEQ ID NO:6;

bovine peptide having the amino acid sequence represented by SEQ IDNO:67;

[Peptide C]

human peptide having the amino acid sequence represented by SEQ ID NO:7,or its amide;

mouse peptide having the amino acid sequence represented by SEQ ID NO:9,or its amide;

rat peptide having the amino acid sequence represented by SEQ ID NO:11,or its amide;

bovine peptide having the amino acid sequence represented by SEQ IDNO:68, or its amide;

[Peptide D]

human peptide having the amino acid sequence represented by SEQ ID NO:8;

mouse peptide having the amino acid sequence represented by SEQ IDNO:10;

rat peptide having the amino acid sequence represented by SEQ ID NO:12;

bovine peptide having the amino acid sequence represented by SEQ IDNO:69;

[Peptide E]

human peptide having the amino acid sequence represented by SEQ IDNO:13;

mouse peptide having the amino acid sequence represented by SEQ IDNO:14;

rat peptide having the amino acid sequence represented by SEQ ID NO:15;

bovine peptide having the amino acid sequence represented by SEQ IDNO:70;

[Peptide F]

human peptide having the amino acid sequence represented by SEQ IDNO:16;

mouse peptide having the amino acid sequence represented by SEQ IDNO:17;

rat peptide having the amino acid sequence represented by SEQ ID NO:18;

bovine peptide having the amino acid sequence represented by SEQ IDNO:71; and the like.

The partial peptide of the present invention may be any peptide so longas it is a partial peptide of the peptide of the present inventiondescribed above. Normally, peptides composed of at least 5 amino acids,preferably at least 10 amino acids are preferred and those furtherhaving activities similar to those of the peptide of the presentinvention are preferred.

The precursor peptide to the peptide of the present invention may be apolypeptide including the peptide of the present invention describedabove, which can produce the peptide of the present invention bycleaving with an appropriate peptidase.

Specifically, proteins, etc. having same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:19 orSEQ ID NO:22 are employed.

The protein having the same or substantially the same amino acidsequence as the amino acid sequence represented by SEQ ID NO:19 or SEQID NO:22 may be any protein derived from any cells of human or otherwarm-blooded animals, e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc. (e.g., retina cells, liver cells,splenocytes, nerve cells, glial cells, cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells,immune cells (e.g., macrophages, T cells, B cells, natural killer cells,mast cells, neutrophils, basophils, eosinophils, monocytes),megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts,osteoclasts, mammary gland cells, hepatocytes or interstitial cells, thecorresponding precursor cells, stem cells, cancer cells, etc., of thesecells), or any tissues where such cells are present, e.g., brain or anyregion of the brain (e.g., retina, olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; or hemocyte typecells or their cultured cells (e.g., MEL, M1, CTLL-2, HT-2, WEHI-3,HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1,Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL,JK-1, CMK, KO-812, MEG-01, etc.). The protein may also be a syntheticprotein.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:19 or SEQ ID NO:22 includes anamino acid sequence having at least about 70% homology, preferably atleast about 80% homology, and more preferably at least about 90%homology, to the amino acid sequence represented by SEQ ID NO:19 or SEQID NO:22.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:19, include:

(i) the amino acid sequence represented by SEQ ID NO:19 (human type);

(ii) the amino acid sequence represented by SEQ ID NO:20 (mouse type);

(iii) the amino acid sequence represented by SEQ ID NO:21 (rat type);

(iv) the amino acid sequence represented by SEQ ID NO:72 (bovine type);

(v) the amino acid sequence represented by SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:72, to which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:72, in which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids arereplaced by other amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

Examples of the amino acid sequence, which has substantially the sameamino acid sequence as that represented by SEQ ID NO:22, include:

(i) the amino acid sequence represented by SEQ ID NO:22 (human type);

(ii) the amino acid sequence represented by SEQ ID NO:23 (mouse type);

(iii) the amino acid sequence represented by SEQ ID NO:24 (rat type);

(iv) the amino acid sequence represented by SEQ ID NO:73 (bovine type);

(v) the amino acid sequence represented by SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24 or SEQ ID NO:73, in which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are deleted;

(vi) the amino acid sequence represented by SEQ ID NO:22, SEQ ID NO:23,SEQ ID NO:24 or SEQ ID NO:73, to which 1 to 15 (preferably 1 to 10, morepreferably 1 to 5, and most preferably 1 to 3) amino acids are added;

(vii) the amino acid sequence represented by SEQ ID NO:22, SEQ ID NO:23or SEQ ID NO:24, in which 1 to 15 (preferably 1 to 10, more preferably 1to 5, and most preferably 1 to 3) amino acids are inserted;

(viii) the amino acid sequence represented by SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24 or SEQ ID NO:73, in which 1 to 15 (preferably 1 to10, more preferably 1 to 5, and most preferably 1 to 3) amino acids arereplaced by other amino acids;

(ix) amino acid sequences in combination of (v) to (viii) above; etc.

The human precursor peptide G having the amino acid sequence representedby SEQ ID NO:19 is the peptide wherein a secretory signal sequence isremoved from the human precursor peptide H having the amino acidsequence represented by SEQ ID NO:22.

The human precursor peptide G having the amino acid sequence representedby SEQ ID NO:20 is the peptide wherein a secretory signal sequence isremoved from the human precursor peptide H having the amino acidsequence represented by SEQ ID NO:23.

The human precursor peptide G having the amino acid sequence representedby SEQ ID NO:21 is the peptide wherein a secretory signal sequence isremoved from the human precursor peptide H having the amino acidsequence represented by SEQ ID NO:24.

The human precursor peptide G having the amino acid sequence representedby SEQ ID NO:72 is the peptide wherein a secretory signal sequence isremoved from the human precursor peptide H having the amino acidsequence represented by SEQ ID NO:73.

The precursor peptide of the present invention may have similaractivities to those of the peptide of the present invention.

The protein (human GPR7) having the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:49,the protein (rat TGR26) having the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:59,the protein (human GPR8) having the same or substantially the same aminoacid sequence as the amino acid sequence represented by SEQ ID NO:84,the protein (bovine GPR7) having the same or substantially the sameamino acid sequence as the amino acid sequence represented by SEQ IDNO:86, the protein (bovine GPR8) having the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO:88 may be any protein derived from any cells of human or otherwarm-blooded animals, e.g., guinea pigs, rats, mice, chicken, rabbits,swine, sheep, bovine, monkeys, etc. (e.g., retina cells, liver cells,splenocytes, nerve cells, glial cells, β cells of pancreas, bone marrowcells, mesangial cells, Langerhans' cells, epidermic cells, epithelialcells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat cells,immune cells (e.g., macrophages, T cells, B cells, natural killer cells,mast cells, neutrophils, basophils, eosinophils, monocytes),megakaryocyte, synovial cells, chondrocytes, bone cells, osteoblasts,osteoclasts, mammary gland cells, hepatocytes or interstitial cells, thecorresponding precursor cells, stem cells, cancer cells, etc., of thesecells), or any tissues where such cells are present, e.g., brain or anyregion of the brain (e.g., retina, olfactory bulb, amygdaloid nucleus,basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,medulla oblongata, cerebellum), spinal cord, hypophysis, stomach,pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow,adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g., largeintestine and small intestine), blood vessel, heart, thymus, spleen,submandibular gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, joint, skeletal muscle, etc.; or hemocyte typecells or their cultured cells (e.g., MEL, M1, CTLL-2, HT-2, WEHI-3,HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1,Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL,JK-1, CMK, KO-812, MEG-01, etc.). The protein may also be a syntheticprotein.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:49 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:49.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:49, include, in addition to the amino acid sequence described above:

(i) the amino acid sequence represented by SEQ ID NO:49, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are deleted;

(ii) the amino acid sequence represented by SEQ ID NO:49, to which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are added;

(iii) the amino acid sequence represented by SEQ ID NO:49, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are inserted;

(iv) the amino acid sequence represented by S SEQ ID NO:49, in which 1to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1to 3) amino acids are replaced by other amino acids;

(v) amino acid sequences in combination of (i) to (iv) above; etc.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:59 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:59.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:59, include, in addition to the amino acid sequence described above:

(i) the amino acid sequence represented by SEQ ID NO:59, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are deleted;

(ii) the amino acid sequence represented by SEQ ID NO:59, to which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are added;

(iii) the amino acid sequence represented by SEQ ID NO:59, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are inserted;

(iv) the amino acid sequence represented by S SEQ ID NO:59, in which 1to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1to 3) amino acids are replaced by other amino acids;

(v) amino acid sequences in combination of (i) to (iv) above; etc.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:84 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, and more preferably at least about 90% homology, to theamino acid sequence represented by SEQ ID NO:84.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:84, include, in addition to the amino acid sequence described above:

(i) the amino acid sequence represented by SEQ ID NO:84, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are deleted;

(ii) the amino acid sequence represented by SEQ ID NO:84, to which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are added;

(iii) the amino acid sequence represented by SEQ ID NO:84, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are inserted;

(iv) the amino acid sequence represented by S SEQ ID NO:84, in which 1to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1to 3) amino acids are replaced by other amino acids;

(v) amino acid sequences in combination of (i) to (iv) above; etc.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:86 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO:86.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:86, include, in addition to the amino acid sequence described above:

(i) the amino acid sequence represented by SEQ ID NO:86, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are deleted;

(ii) the amino acid sequence represented by SEQ ID NO:86, to which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are added;

(iii) the amino acid sequence represented by SEQ ID NO:86, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are inserted;

(iv) the amino acid sequence represented by S SEQ ID NO:86, in which 1to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1to 3) amino acids are replaced by other amino acids;

(v) amino acid sequences in combination of (i) to (iv) above; etc.

The amino acid sequence which has substantially the same amino acidsequence as that represented by SEQ ID NO:88 includes an amino acidsequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to the amino acid sequencerepresented by SEQ ID NO:88.

In particular, examples of the amino acid sequence, which hassubstantially the same amino acid sequence as that represented by SEQ IDNO:88, include, in addition to the amino acid sequence described above:

(i) the amino acid sequence represented by SEQ ID NO:88, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are deleted;

(ii) the amino acid sequence represented by SEQ ID NO:88, to which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are added;

(iii) the amino acid sequence represented by SEQ ID NO:88, in which 1 to15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to3) amino acids are inserted;

(iv) the amino acid sequence represented by S SEQ ID NO:88, in which 1to 15 (preferably 1 to 10, more preferably 1 to 5, and most preferably 1to 3) amino acids are replaced by other amino acids;

(v) amino acid sequences in combination of (i) to (iv) above; etc.

The partial peptide of human GPR7, rat TGR26, human GPR8, bovine GPR7 orbovine GPR8 (hereinafter merely referred to as GPR7 collectively) may beany peptide, so long as it is a partial peptide usable in the methodsfor screening pharmaceuticals, etc. later described, but, a partialpeptide capable of binding to the peptide of the present invention, apartial peptide containing the corresponding amino acid sequence in thearea outside the cell membrane, etc. are preferably employed.

The peptide of the present invention, its partial peptide or itsprecursor peptide, especially the peptide of the present invention alsoincludes a peptide wherein the N-terminal amino acid residue isbrominated. Preferred examples of the N-terminal amino acid residue aretryptophan residue (Trp), etc.

Specifically, a peptide containing the amino acid sequence selected fromSEQ ID NO:1 to SEQ ID NO:12, SEQ ID NO:19 to SEQ ID NO:21, SEQ ID NO:66to SEQ ID NO:69 and SEQ ID NO:72, etc., wherein the N-terminaltryptophan residue (Trp) is brominated, is employed. Among thesepeptides, preferably employed is a peptide containing the amino acidsequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:66 or SEQ ID NO:67, whereinthe N-terminal tryptophan residue (Trp) is brominated. The position tobe brominated is not particularly limited but the tryptophan residue(Trp) at the 6-position is preferred.

More specifically, a peptide containing the amino acid sequence selectedfrom SEQ ID NO:1 to SEQ ID NO:12, SEQ ID NO:19 to SEQ ID NO:21, SEQ IDNO:66 to SEQ ID NO:69 and SEQ ID NO:72, wherein the N-terminaltryptophan residue (Trp) is 6-brominated, is preferably employed. Amongthem, preferably employed is a peptide containing the amino acidsequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ. ID NO:66 or SEQ ID NO:67, whereinthe N-terminal tryptophan residue (Trp) is 6-brominated.

The peptide of the present invention, its partial peptide or itsprecursor peptide (hereinafter sometimes merely referred to as thepeptide of the present invention), and GPR7 or its partial peptide(hereinafter sometimes merely referred to as GPR7) are represented inaccordance with the conventional way of describing peptides, that is,the N-terminus (amino terminus) at the left hand and the C-terminus(carboxyl terminus) at the right hand.

In the peptide of the present invention or GPR7, the C-terminus may beany of a carboxyl group (—COOH), a carboxylate (—COO⁻), an amide(—CONH₂) or an ester (—COOR).

Examples of the ester group shown by R include a C₁₋₆ alkyl group suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; a C₃₋₈ cycloalkylgroup such as cyclopentyl, cyclohexyl, etc.; a C₆₋₁₂ aryl group such asphenyl, α-naphthyl, etc.; a C₇₋₁₄ aralkyl group such as aphenyl-C₁₋₂-alkyl group, e.g., benzyl, phenethyl, etc., or anα-naphthyl-C₁₋₂-alkyl group such as α-naphthylmethyl, etc.; and thelike. In addition, pivaloyloxymethyl or the like, which is used widelyas an ester for oral administration, may also be used.

Where the peptide of the present invention or GPR7 contains a carboxylgroup (or a carboxylate) at a position other than the C-terminus, it maybe amidated or esterified and such an amide or ester is also includedwithin the peptide of the present invention. The ester group may be thesame group as that described with respect to the C-terminus describedabove.

Furthermore, the peptide of the present invention or GPR7 includespeptides, wherein the amino group at the N-terminal amino acid residue(e.g., methionine residue) is protected with a protecting group (e.g., aC₁₋₆ acyl group such as a C₂₋₆ alkanoyl group, e.g., formyl group,acetyl group, etc.); those wherein the N-terminal region is cleaved invivo and the glutamyl group thus formed is pyroglutaminated; thosewherein a substituent (e.g., —OH, —SH, amino group, imidazole group,indole group, guanidino group, etc.) on the side chain of an amino acidin the molecule is protected with a suitable protecting group (e.g., aC₁₋₆ acyl group such as a C₂₋₆ alkanoyl group, e.g., formyl group,acetyl group, etc.), or conjugated proteins such as glycoproteins boundto sugar chains.

For salts of the peptide of the present invention or GPR7, preferred aresalts with physiologically acceptable acids (e.g., inorganic acids ororganic acids) or bases (e.g., alkali metal salts), etc., especiallyphysiologically acceptable acid addition salts. Examples of such saltsinclude salts with, for example, inorganic acids (e.g., hydrochloricacid, phosphoric acid, hydrobromic acid, sulfuric acid); salts withorganic acids (e.g., acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonicacid) and the like. Hereinafter, the peptide of the present invention orGPR7 is used to include these salts as well.

The peptide of the present invention or GPR7 may be manufactured by apublicly known method used to purify a peptide from human or otherwarm-blooded animal cells or tissues described above, or by culturing atransformant that contains the DNA encoding the peptide, as will belater described. Furthermore, the peptide of the present invention orGPR7 may also be manufactured by the methods for synthesizing peptidesor by modifications thereof, which will also be described hereinafter.

Where the peptide of the present invention or GPR7 is manufactured fromhuman or mammalian tissues or cells, human or mammalian tissues or cellsare homogenized, then extracted with an acid or the like, and theextract is isolated and purified by a combination of chromatographytechniques such as reverse phase chromatography, ion exchangechromatography, and the like.

To synthesize the peptide of the present invention or GPR7 or amidesthereof, commercially available resins that are used for peptidesynthesis may be used. Examples of such resins include chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAMresin, 4-hydroxymethylmehtylphenyl acetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Usingthese resins, amino acids in which α-amino groups and functional groupson the side chains are appropriately protected are condensed on theresin in the order of the sequence of the objective peptide according tovarious condensation methods publicly known in the art. At the end ofthe reaction, the peptide is cut out from the resin and at the sametime, the protecting groups are removed. Then, intramolecular disulfidebond-forming reaction is performed in a highly diluted solution toobtain the objective peptide of the present invention or GPR7, or amidesthereof.

For condensation of the protected amino acids described above, a varietyof activation reagents for peptide synthesis may be used, andcarbodiimides are particularly preferable. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide, etc. For activation bythese reagents, the protected amino acids in combination with aracemization inhibitor (e.g., HOBt, HOOBt) are added directly to theresin, or the protected amino acids are previously activated in the formof symmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

Solvents suitable for use to activate the protected amino acids orcondense with the resin may be chosen from solvents known to be usablefor peptide condensation reactions. Examples of such solvents are acidamides such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpyrrolidone, etc.; halogenated hydrocarbons such as methylenechloride, chloroform, etc.; alcohols such as trifluoroethanol, etc.;sulfoxides such as dimethylsulfoxide, etc.; ethers such as pyridine,dioxan, tetrahydrofuran, etc.; nitriles such as acetonitrile,propionitrile, etc.; esters such as methyl acetate, ethyl acetate, etc.;and appropriate mixtures of these solvents. The reaction temperature isappropriately chosen from the range known to be applicable to peptidebinding reactions and is usually selected in the range of approximately−20° C. to 50° C. The activated amino acid derivatives are usedgenerally in an excess of 1.5 to 4 times. The condensation is examinedby a test using the ninhydrin reaction; when the condensation isinsufficient, the condensation can be completed by repeating thecondensation reaction without removal of the protecting groups. When thecondensation is yet insufficient even after repeating the reaction,unreacted amino acids are acetylated with acetic anhydride oracetylimidazole so as not to affect the following reactions.

Examples of the protecting groups for amino groups of the startingcompounds include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl,diphenylphosphinothioyl, Fmoc, etc.

A carboxyl group can be protected by, e.g., alkyl esterification (e.g.,esterification of linear, branched or cyclic alkyl moiety such asmethyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, etc.), aralkyl esterification(e.g., esterification in the form of benzyl ester, 4-nitrobenzyl ester,4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl ester, etc.),phenacyl esterification, benzyloxycarbonyl hydrazidation,t-butoxycarbonyl hydrazidation, trityl hydrazidation, or the like.

The hydroxyl group of serine can be protected through, for example, itsesterification or etherification. Examples of groups appropriately usedfor the esterification include a lower alkanoyl group, such as acetylgroup, an aroyl group such as benzoyl group, and a group derived fromcarbonic acid such as benzyloxycarbonyl group, ethoxycarbonyl group,etc. Examples of a group appropriately used for the etherificationinclude benzyl group, tetrahydropyranyl group, t-butyl group, etc.

Examples of groups for protecting the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl, etc.

Examples of groups used to protect the imidazole moiety of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

Examples of the activated carboxyl groups in the starting compoundsinclude the corresponding acid anhydrides, azides, activated esters(esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, HOBt)). As the activated aminoacids, in which the amino groups are activated in the starting material,the corresponding phosphoric amides are employed.

To eliminate (split off) the protecting groups, there are used catalyticreduction under hydrogen gas flow in the presence of a catalyst such asPd-black or Pd-carbon; an acid treatment with anhydrous hydrogenfluoride, methanesulfonic acid, trifluoromethane-sulfonic acid ortrifluoroacetic acid, or a mixture solution of these acids; a treatmentwith a base such as diisopropylethylamine, triethylamine, piperidine orpiperazine; and reduction with sodium in liquid ammonia. The eliminationof the protecting group by the acid treatment described above is carriedout generally at a temperature of approximately −20° C. to 40° C. In theacid treatment, it is efficient to add a cation scavenger such asanisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide,1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, 2,4-dinitrophenylgroup known as the protecting group for the imidazole of histidine isremoved by a treatment with thiophenol. Formyl group used as theprotecting group of the indole of tryptophan is eliminated by theaforesaid acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by a treatment with an alkali such as adilute sodium hydroxide solution, dilute ammonia, etc.

Protection of functional groups that should not be involved in thereaction of the starting materials, protecting groups, elimination ofthe protecting groups and activation of functional groups involved inthe reaction may be appropriately selected from publicly known groupsand publicly known means.

In another method for obtaining the amides of the peptide of the presentinvention or GPR7, for example, the α-carboxyl group of the carboxyterminal amino acid is first protected by amidation; the peptide chainis then extended from the amino group side to a desired length.Thereafter, a peptide in which only the protecting group of theN-terminal α-amino group in the peptide chain has been eliminated fromthe peptide and a peptide in which only the protecting group of theC-terminal carboxyl group has been eliminated are prepared. The twopeptides are condensed in a mixture of the solvents described above. Thedetails of the condensation reaction are the same as described above.After the protected peptide obtained by the condensation is purified,all the protecting groups are eliminated by the method described aboveto give the desired crude peptide. This crude peptide is purified byvarious known purification means. Lyophilization of the major fractiongives the amides of the desired peptide of the present invention orGPR7.

To prepare the esterified form of the peptide of the present inventionor GPR7, for example, the α-carboxyl group of the carboxy terminal aminoacid is condensed with a desired alcohol to prepare the amino acidester, which is followed by procedure similar to the preparation of theamidated form of the peptide of the present invention or GPR7 to givethe desired ester form of the peptide of the present invention or GPR7.

The partial peptide of the peptide of the present invention or GPR7 canbe manufactured by publicly known methods for peptide synthesis, or thepartial peptide of GPR7 can be manufactured by cleaving GPR7 with anappropriate peptidase. For the methods for peptide synthesis, forexample, either solid phase synthesis or liquid phase synthesis may beused. That is, the partial peptide or amino acids that can construct thepeptide of the present invention or the partial peptide of GPR7 arecondensed with the remaining part. Where the product contains protectinggroups, these protecting groups are removed to give the desired peptide.Publicly known methods for condensation and elimination of theprotecting groups are described in (1)-(5) below.

-   (1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, Interscience    Publishers, New York (1966)-   (2) Schroeder & Luebke: The Peptide, Academic Press, New York (1965)-   (3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basics    and experiments of peptide synthesis), published by Maruzen Co.    (1975)-   (4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza    (Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of    Proteins) IV, 205 (1977)-   (5) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu (A sequel to    Development of Pharmaceuticals), Vol. 14, Peptide Synthesis,    published by Hirokawa Shoten

After completion of the reaction, the peptide of the present invention,GPR7 or a partial peptide thereof may be purified and isolated by acombination of conventional purification methods such as solventextraction, distillation, column chromatography, liquid chromatographyand recrystallization to give the partial peptide of the presentinvention. When the peptide of the present invention or the partialpeptide of GPR7 obtained by the above methods is in a free form, thepeptide can be converted into an appropriate salt by a publicly knownmethod; when the product is obtained in the form of a salt, it can beconverted into a free form or other salts by a publicly known method orits modification.

The polynucleotide encoding the peptide of the present invention or GPR7may be any polynucleotide so long as it contains the base sequence (DNAor RNA, preferably DNA) encoding the peptide of the present invention orGPR7 described above. Such a polynucleotide may also be any one of DNAencoding the peptide of the present invention or GPR7, RNA such as mRNA,etc., and may be double-stranded or single-stranded. Where thepolynucleotide is double-stranded, it may be double-stranded DNA,double-stranded RNA or DNA:RNA hybrid. Where the polynucleotide issingle-stranded, it may be a sense strand (i.e., a coding strand) or anantisense strand (i.e., a non-coding strand).

Using the polynucleotide encoding the peptide of the present inventionor GPR7, mRNA of the peptide of the present invention or GPR7 can bequantified by, for example, the publicly known method published inseparate volume of Jikken Igaku 15 (7) “New PCR and its application”(1997), or by its modifications.

The DNA encoding the peptide of the present invention or GPR7 may be anyDNA, as long as it contains a base sequence encoding the peptide of thepresent invention or GPR7 described above. The DNA may also be any ofgenomic DNA, genomic DNA library, cDNA derived from the cells andtissues described above, cDNA library derived from the cells and tissuesdescribed above and synthetic DNA.

The vector to be used for the library may be any of bacteriophage,plasmid, cosmid and phagemid. The DNA may also be directly amplified byreverse transcriptase polymerase chain reaction (hereinafter abbreviatedas RT-PCR) using the total RNA or mRNA fraction prepared from the cellsand tissues described above.

The DNA encoding the peptide of the present invention may be any DNA, solong as it is a DNA having a base sequence hybridizable to the basesequence represented by any sequence identification number of SEQ IDNO:25 to SEQ ID NO:42 and SEQ ID NO:74 to SEQ ID NO:79 under highlystringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of the peptide of the presentinvention.

Specific examples of the DNA hybridizable to the base sequencerepresented by any sequence identification number of SEQ ID NO:25 to SEQID NO:42 and SEQ ID NO:74 to SEQ ID NO:79 under highly stringentconditions include DNAs containing a base sequence having at least about70% homology, preferably at least about 80% homology, more preferably atleast about 90% homology and the most preferably at least about 95%homology, to the base sequence represented by any sequenceidentification number of SEQ ID NO:25 to SEQ ID NO:42 and SEQ ID NO:74to SEQ ID NO:79.

The hybridization can be carried out by publicly known methods or bymodifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989). A commercially available library may also beused according to the instructions of the attached manufacturer'sprotocol. Preferably, the hybridization can be carried out under highlystringent conditions.

The highly stringent conditions used herein are, for example, those in asodium concentration at about 19 to 40 mM, preferably about 19 to 20 mMat a temperature of about 50 to 70° C., preferably about 60 to 65° C. Inparticular, hybridization conditions in a sodium concentration of about19 mM at a temperature of about 65° C. are most preferred.

More specifically,

(i) for the DNA encoding human peptide A containing the amino acidsequence represented by SEQ ID NO:1, there are employed a DNA containingthe base sequence represented by SEQ ID NO:25, etc.;

(ii) for the DNA encoding mouse peptide A containing the amino acidsequence represented by SEQ ID NO:2, there are employed a DNA containingthe base sequence represented by SEQ ID NO:26, etc.;

(iii) for the DNA encoding rat peptide A containing the amino acidsequence represented by SEQ ID NO:3, there are employed a DNA containingthe base sequence represented by SEQ ID NO:27, etc.;

(iv) for the DNA encoding human peptide B containing the amino acidsequence represented by SEQ ID NO:4, there are employed a DNA containingthe base sequence represented by SEQ ID NO:28, etc.;

(v) for the DNA encoding mouse peptide B containing the amino acidsequence represented by SEQ ID NO:5, there are employed a DNA containingthe base sequence represented by SEQ ID NO:29, etc.;

(vi) for the DNA encoding rat peptide B containing the amino acidsequence represented by SEQ ID NO:6, there are employed a DNA containingthe base sequence represented by SEQ ID NO:30, etc.;

(vii) for the DNA encoding human peptide C containing the amino acidsequence represented by SEQ ID NO:7, there are employed a DNA containingthe base sequence represented by SEQ ID NO:31, etc.;

(viii) for the DNA encoding human peptide D containing the amino acidsequence represented by SEQ ID NO:8, there are employed a DNA containingthe base sequence represented by SEQ ID NO:32, etc.;

(ix) for the DNA encoding mouse peptide C containing the amino acidsequence represented by SEQ ID NO:9, there are employed a DNA containingthe base sequence represented by SEQ ID NO:33, etc.;

(x) for the DNA encoding mouse peptide D containing the amino acidsequence represented by SEQ ID NO:10, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:34, etc.;

(xi) for the DNA encoding rat peptide C containing the amino acidsequence represented by SEQ ID NO:11, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:35, etc.;

(xii) for the DNA encoding rat peptide D containing the amino acidsequence represented by SEQ ID NO:12, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:36, etc.;

(xiii) for the DNA encoding human peptide E containing the amino acidsequence represented by SEQ ID NO:13, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:37, etc.;

(xiv) for the DNA encoding mouse peptide E containing the amino acidsequence represented by SEQ ID NO:14, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:38, etc.;

(xv) for the DNA encoding rat peptide E containing the amino acidsequence represented by SEQ ID NO:15, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:39, etc.;

(xvi) for the DNA encoding human peptide F containing the amino acidsequence represented by SEQ ID NO:16, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:40, etc.;

(xvii) for the DNA encoding mouse peptide F containing the amino acidsequence represented by SEQ ID NO:17, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:41, etc.;

(xviii) for the DNA encoding rat peptide F containing the amino acidsequence represented by SEQ ID NO:18, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:42, etc.;

(xix) for the DNA encoding bovine peptide A containing the amino acidsequence represented by SEQ ID NO:66, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:74, etc.;

(xx) for the DNA encoding bovine peptide B containing the amino acidsequence represented by SEQ ID NO:67, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:75, etc.;

(xxi) for the DNA encoding bovine peptide C containing the amino acidsequence represented by SEQ ID NO:68, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:76, etc.;

(xxii) for the DNA encoding bovine peptide D containing the amino acidsequence represented by SEQ ID NO:69, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:77, etc.;

(xxiii) for the DNA encoding bovine peptide E containing the amino acidsequence represented by SEQ ID NO:70, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:78, etc.;

(xxvi) for the DNA encoding bovine peptide F containing the amino acidsequence represented by SEQ ID NO:71, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:79; etc.

The DNA encoding the partial peptide of the present invention may be anyDNA, as long as it contains a base sequence encoding the partial peptideof the present invention described above. The DNA may also be any ofgenomic DNA, genomic DNA library, cDNA derived from the cells andtissues described above, cDNA library derived from the cells and tissuesdescribed above and synthetic DNA.

The DNA encoding the partial peptide of the present invention is, forexample, a DNA having a partial base sequence of the DNA having the basesequence represented by any sequence identification number of SEQ IDNO:25 to SEQ ID NO:42 and SEQ ID NO:74 to SEQ ID NO:79, or a DNA havinga base sequence hybridizable to the base sequence represented by anysequence identification number of SEQ ID NO:25 to SEQ ID NO:42 and SEQID NO:74 to SEQ ID NO:79 under highly stringent conditions and encodinga peptide having the activities substantially equivalent to those of thepeptide of the present invention.

The DNA hybridizable to the base sequence represented by any sequenceidentification number of SEQ ID NO:25 to SEQ ID NO:42 and SEQ ID NO:74to SEQ ID NO:79 has the same significance as described above.

With respect to the hybridization under high stringent conditions, thesame procedures as described above apply.

The DNA encoding the precursor peptide of the present invention may beany DNA, so long as it is a DNA having a base sequence hybridizable tothe base sequence represented by SEQ ID NO:43 or SEQ ID NO:46under-highly stringent conditions and encoding a peptide having theactivities substantially equivalent to those of the precursor of thepresent invention.

The DNA hybridizable to the base sequence represented by SEQ ID NO:43 orSEQ ID NO:46 under highly stringent conditions includes a DNA containinga base sequence having at least about 70% homology, preferably at leastabout 80% homology, more preferably at least about 90% homology, andmost preferably at least about 95% homology, to a base sequencerepresented by any sequence identification number of SEQ ID NO:43 or SEQID NO:46.

With respect to the hybridization under high stringent conditions, thesame procedures the same conditions as described above apply.

More specifically,

(i) for the DNA encoding human GPR7 ligand precursor G containing theamino acid sequence represented by SEQ ID NO:19, there are employed aDNA containing the base sequence represented by SEQ ID NO:43, etc.;

(ii) for the DNA encoding mouse GPR7 ligand precursor G containing theamino acid sequence represented by SEQ ID NO:20, there are employed aDNA containing the base sequence represented by SEQ ID NO:44, etc.;

(iii) for the DNA encoding rat GPR7 ligand precursor G containing theamino acid sequence represented by SEQ ID NO:21, there are employed aDNA containing the base sequence represented by SEQ ID NO:45, etc.;

(iv) for the DNA encoding bovine GPR7 ligand precursor G containing theamino acid sequence represented by SEQ ID NO:72, there are employed aDNA containing the base sequence represented by SEQ ID NO:80, etc.;

(v) for the DNA encoding mouse GPR7 ligand precursor H containing theamino acid sequence represented by SEQ ID NO:22, there are employed aDNA containing the base sequence represented by SEQ ID NO:46, etc.;

(vi) for the DNA encoding mouse GPR7 ligand precursor H containing theamino acid sequence represented by SEQ ID NO:23, there are employed aDNA containing the base sequence represented by SEQ ID NO:47, etc.;

(vii) for the DNA encoding rat GPR7 ligand precursor H containing theamino acid sequence represented by SEQ ID NO:24, there are employed aDNA containing the base sequence represented by SEQ ID NO:48, etc.;

(viii) for the DNA encoding bovine GPR7 ligand precursor H containingthe amino acid sequence represented by SEQ ID NO:73, there are employeda DNA containing the base sequence represented by SEQ ID NO:81, etc.

The polynucleotide comprising a part of the base sequence of the DNAencoding the peptide or partial peptide of the present invention, or apart of the base sequence complementary to the DNA is used to mean toembrace not only the DNA encoding the partial peptide of the presentinvention but also RNA.

According to the present invention, antisense polynucleotides (nucleicacids) that can inhibit the replication or expression of genes for thepeptide of the present invention can be designed and synthesized basedon the base sequence information of the cloned or determined DNAencoding the peptide of the present invention. Such a polynucleotide(nucleic acid) is capable of hybridizing to RNA of genes for the peptideof the present invention to inhibit the synthesis or function of saidRNA or capable of modulating or controlling the expression of genes forthe peptide of the present invention via interaction with RNA associatedwith the peptide of the present invention. Polynucleotides complementaryto the selected sequences of RNA associated with the peptide of thepresent invention and polynucleotides specifically hybridizable to theRNA associated with the peptide of the present invention are useful inmodulating or controlling the expression of genes for the peptide of thepresent invention in vivo and in vitro, and useful for the treatment ordiagnosis of diseases, etc. The term “corresponding” is used to meanhomologous to or complementary to a particular sequence of thenucleotide, base sequence or nucleic acid, including the genes. The term“corresponding” between nucleotides, base sequences or nucleic acids andpeptides (proteins) usually refer to amino acids of a peptide (protein)under the order derived from the sequence of nucleotides (nucleic acids)or their complements. In the genes for the peptide of the presentinvention, the 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ enduntranslated region, polypeptide translation initiation codon, proteincoding region, ORF translation initiation codon, 3′ end untranslatedregion, 3′ end palindrome region, and 3′ end hairpin loop, may beselected as preferred target regions, though any other region may beselected as a target in the genes for the peptide of the presentinvention.

The relationship between the targeted nucleic acids and thepolynucleotides complementary to at least a part of the target,specifically the relationship between the target and the polynucleotideshybridizable to the target, can be denoted to be “antisense”. Examplesof the antisense polynucleotides include polynucleotides containing2-deoxy-D-ribose, polynucleotides containing D-ribose, any other type ofpolynucleotides which are N-glycosides of a purine or pyrimidine base,or other polymers containing non-nucleotide backbones (e.g., proteinnucleic acids and synthetic sequence-specific nucleic acid polymerscommercially available) or other polymers containing nonstandardlinkages (provided that the polymers contain nucleotides having such aconfiguration that allows base pairing or base stacking, as is found inDNA or RNA), etc. The antisense polynucleotides may be double-strandedDNA, single-stranded DNA, single-stranded RNA or a DNA:RNA hybrid, andmay further include unmodified polynucleotides (or unmodifiedoligonucleotides), those with publicly known types of modifications, forexample, those with labels known in the art, those with caps, methylatedpolynucleotides, those with substitution of one or more naturallyoccurring nucleotides by their analogue, those with intramolecularmodifications of nucleotides such as those with uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.) and those with charged linkages or sulfur-containinglinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosehaving side chain groups such as proteins (nucleases, nucleaseinhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.),saccharides (e.g., monosaccharides, etc.), those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylating agents, those with modified linkages (e.g., aanomeric nucleic acids, etc.), and the like. Herein the terms“nucleoside”, “nucleotide” and “nucleic acid” are used to refer tomoieties that contain not only the purine and pyrimidine bases, but alsoother heterocyclic bases, which have been modified. Such modificationsmay include methylated purines and pyrimidines, acylated purines andpyrimidines and other heterocyclic rings. Modified nucleotides andmodified nucleotides also include modifications on the sugar moiety,wherein, for example, one or more hydroxyl groups may optionally besubstituted with a halogen atom(s), an aliphatic group(s), etc., or maybe converted into the corresponding functional groups such as ethers,amines, or the like.

The antisense polynucleotide (nucleic acid) of the present invention isRNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of themodified nucleic acid are, but not limited to, sulfur and thiophosphatederivatives of nucleic acids and those resistant to degradation ofpolynucleoside amides or oligonucleoside amides. The antisense nucleicacids of the present invention can be modified preferably based on thefollowing design, that is, by increasing the intracellular stability ofthe antisense nucleic acid, increasing the cellular permeability of theantisense nucleic acid, increasing the affinity of the nucleic acid tothe targeted sense strand to a higher level, or minimizing the toxicity,if any, of the antisense nucleic acid.

Many of such modifications are known in the art, as disclosed in J.Kawakami, et al., Pharm. Tech. Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp.395, 1992; S. T. Crooke, et al. ed., Antisense Research andApplications, CRC Press, 1993; etc.

The antisense nucleic acid of the present invention may contain alteredor modified sugars, bases or linkages. The antisense nucleic acid mayalso be provided in a specialized form such as liposomes, microspheres,or may be applied to gene therapy, or may be provided in combinationwith attached moieties. Such attached moieties include polycations suchas polylysine that act as charge neutralizers of the phosphate backbone,or hydrophobic moieties such as lipids (e.g., phospholipids,cholesterols, etc.) that enhance the interaction with cell membranes orincrease uptake of the nucleic acid. Preferred examples of the lipids tobe attached are cholesterols or derivatives thereof (e.g., cholesterylchloroformate, cholic acid, etc.). These moieties may be attached to thenucleic acid at the 3′ or 5′ ends thereof and may also be attachedthereto through a base, sugar, or intramolecular nucleoside linkage.Other moieties may be capping groups specifically placed at the 3′ or 5′ends of the nucleic acid to prevent degradation by nucleases such asexonuclease, RNase, etc. Such capping groups include, but are notlimited to, hydroxyl protecting groups known in the art, includingglycols such as polyethylene glycol, tetraethylene glycol and the like.

The inhibitory action of the antisense nucleic acid can be examinedusing the transformant of the present invention, the gene expressionsystem of the present invention in vivo and in vitro, or the translationsystem of the peptide of the present invention in vivo and in vitro. Thenucleic acid can be applied to cells by a variety of publicly knownmethods.

The DNA encoding human GPR7 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:50, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:50 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of human GPR7having the base sequence represented by SEQ ID NO:49, or the like.

The DNA hybridizable to the base sequence represented by SEQ ID NO:50under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:50, or the like.

The DNA encoding rat TGR26 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:60, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:60 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of rat TGR26having the base sequence represented by SEQ ID NO:59, or the like.

The DNA hybridizable to the base sequence represented by SEQ ID NO:60under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:60, or the like.

The DNA encoding human GPR8 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:85, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:85 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of human GPR8having the base sequence represented by SEQ ID NO:66, or the like.

The DNA hybridizable to the base sequence represented by SEQ ID NO:85under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:85, or the like.

The DNA encoding bovine GPR7 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:87, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:87 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of bovine GPR7having the base sequence represented by SEQ ID NO:86, or the like.

The DNA hybridizable to the base sequence represented by SEQ ID NO:87under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:87, or the like.

The DNA encoding bovine GPR8 may be any DNA, as far as it is a DNAcontaining the base sequence represented by, e.g., SEQ ID NO:89, a DNAhaving a base sequence hybridizable to the base sequence represented bySEQ ID NO:89 under high stringent conditions and encoding a proteinhaving the activities substantially equivalent to those of bovine GPR8having the base sequence represented by SEQ ID NO:88, or the like.

The DNA hybridizable to the base sequence represented by SEQ ID NO:89under highly stringent conditions includes a DNA containing a basesequence having at least about 70% homology, preferably at least about80% homology, more preferably at least about 90% homology, and mostpreferably at least about 95% homology, to a base sequence representedby SEQ ID NO:89, or the like.

The hybridization can be carried out by publicly known methods or bymodifications of these methods, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989), etc. A commercially available library may alsobe used according to the instructions of the attached manufacturer'sprotocol. Preferably, the hybridization can be carried out under highlystringent conditions.

The highly stringent conditions used herein are, for example, those in asodium concentration at about 19 to 40 mM, preferably about 19 to 20 mMat a temperature of about 50 to 70° C., preferably about 60 to 65° C. Inparticular, hybridization conditions in a sodium concentration of about19 mM at a temperature of about 65° C. are most preferred.

More specifically, for the DNA encoding human GPR7 containing the aminoacid sequence represented by SEQ ID NO:49, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:50, etc.; for theDNA encoding rat TGR26 containing the amino acid sequence represented bySEQ ID NO:59, there are employed a DNA containing the base sequencerepresented by SEQ ID NO:60, etc.; for the DNA encoding human GPR8containing the amino acid sequence represented by SEQ ID NO:84, thereare employed a DNA containing the base sequence represented by SEQ IDNO:85, etc.; for the DNA encoding bovine GPR7 containing the amino acidsequence represented by SEQ ID NO:86, there are employed a DNAcontaining the base sequence represented by SEQ ID NO:87, etc.; and, forthe DNA encoding bovine GPR8 containing the amino acid sequencerepresented by SEQ ID NO:88, there are employed a DNA containing thebase sequence represented by SEQ ID NO:89, etc.

The DNA encoding the partial peptide of GPR7 may be any DNA, as long asit contains a base sequence encoding the partial peptide of GPR7described above. The DNA may also be any of genomic DNA, genomic DNAlibrary, cDNA derived from the cells and tissues described above, cDNAlibrary derived from the cells and tissues described above and syntheticDNA.

The DNA encoding the partial peptide of human GPR7 is, for example, aDNA having a partial base sequence of the DNA having the base sequencerepresented by SEQ ID NO:50, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:50 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of human GPR7.

The DNA hybridizable to the base sequence represented by SEQ ID NO:50has the same significance as described above.

The DNA encoding the partial peptide of rat TGR26 is, for example, a DNAhaving a partial base sequence of the DNA having the base sequencerepresented by SEQ ID NO:60, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:60 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of rat TGR26.

The DNA hybridizable to the base sequence represented by SEQ ID NO:60has the same significance as described above.

The DNA encoding the partial peptide of human GPR8 may be any DNA, aslong as it contains a base sequence encoding the partial peptide ofhuman GPR8 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

The DNA encoding the partial peptide of human GPR8 is, for example, aDNA having a partial base sequence of the DNA having the base sequencerepresented by SEQ ID NO:85, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:85 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of human GPR8.

The DNA hybridizable to the base sequence represented by SEQ ID NO:85has the same significance as described above.

The DNA encoding the partial peptide of bovine GPR7 may be any DNA, aslong as it contains a base sequence encoding the partial peptide ofbovine GPR7 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

The DNA encoding the partial peptide of bovine GPR7 is, for example, aDNA having a partial base sequence of the DNA having the base sequencerepresented by SEQ ID NO:87, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:87 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of bovine GPR7.

The DNA hybridizable to the base sequence represented by SEQ ID NO:87has the same significance as described above.

The DNA encoding the partial peptide of bovine GPR8 may be any DNA, aslong as it contains a base sequence encoding the partial peptide ofbovine GPR8 described above. The DNA may also be any of genomic DNA,genomic DNA library, cDNA derived from the cells and tissues describedabove, cDNA library derived from the cells and tissues described aboveand synthetic DNA.

The DNA encoding the partial peptide of bovine GPR8 is, for example, aDNA having a partial base sequence of the DNA having the base sequencerepresented by SEQ ID NO:89, or a DNA having a base sequencehybridizable to the base sequence represented by SEQ ID NO:89 underhighly stringent conditions and encoding a peptide having the activitiessubstantially equivalent to those of bovine GPR8.

The DNA hybridizable to the base sequence represented by SEQ ID NO:89has the same significance as described above.

With respect to the hybridization under high stringent conditions, thesame procedures as described-above apply.

The DNA encoding the peptide of the present invention or GPR7 may belabeled by publicly known methods. Specific examples include thoselabeled with an isotope, those labeled with fluorescence (labeling with,e.g., fluorescein, etc.), those biotinated, those labeled with enzyme,etc.

For cloning of the DNA that fully encodes the peptide of the presentinvention or GPR7, the DNA may be either amplified by publicly known PCRusing synthetic DNA primers containing a part of the base sequence ofthe peptide of the present invention or GPR7, or the DNA inserted intoan appropriate vector can be selected by hybridization with a labeledDNA fragment or synthetic DNA that encodes a part or entire region ofthe peptide of the present invention or GPR7. The hybridization can becarried out, for example, according to the method described in MolecularCloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989),etc. The hybridization may also be performed using commerciallyavailable library in accordance with the protocol described in theattached instructions.

Conversion of the base sequence of DNA can be made by publicly knownmethods such as the ODA-LA PCR method, the Gapped duplex method or theKunkel method, or modifications thereof, by using a publicly known kitavailable as Mutan™-super Express Km (manufactured by TaKaRa Shuzo Co.,Ltd., trademark), Mutan™-K (manufactured by TaKaRa Shuzo Co., Ltd.,trademark), etc.

The cloned peptide-encoding DNA can be used as it is, depending uponpurpose or, if desired, after digestion with a restriction enzyme orafter addition of a linker thereto. The DNA may contain ATG as atranslation initiation codon at the 5′ end thereof and TAA, TGA or TAGas a translation termination codon at the 3′ end thereof. Thesetranslation initiation and termination codons may also be added by usingan appropriate synthetic DNA adapter.

The expression vector of the peptide of the present invention or GPR7can be manufactured, for example, by (a) excising the desired DNAfragment from the DNA encoding the peptide of the present invention orGPR7, (b) and then ligating the DNA fragment with an appropriateexpression vector downstream a promoter in the vector.

Examples of the vector include plasmids derived form E. coli (e.g.,pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis(e.g., pUB110, pTP5, pC194), plasmids derived from yeast (e.g., pSH19,pSH15), bacteriophages such as λ phage, etc., animal viruses such asretrovirus, vaccinia virus, baculovirus, etc. as well as pA1-11, pXT1,pRc/CMV, pRc/RSV, pcDNAI/Neo, etc.

The promoter used in the present invention may be any promoter if itmatches well with a host to be used for gene expression. In the case ofusing animal cells as the host, examples of the promoter include SRαpromoter, SV40 promoter, HIV•LTR promoter, CMV promoter, HSV-TKpromoter, etc.

Among them, CMV (cytomegalovirus) promoter or SRα promoter is preferablyused. Where the host is bacteria of the genus Escherichia, preferredexamples of the promoter include trp promoter, lac promoter, recApromoter, λPL promoter, lpp promoter, T7 promoter, etc. In the case ofusing bacteria of the genus Bacillus as the host, preferred example ofthe promoter are SPO1 promoter, SPO2 promoter and penP promoter. Whenyeast is used as the host, preferred examples of the promoter are PHO5promoter, PGK promoter, GAP promoter and ADH promoter. When insect cellsare used as the host, preferred examples of the promoter includepolyhedrin promoter, P10 promoter, etc.

In addition to the foregoing examples, the expression vector may furtheroptionally contain an enhancer, a splicing signal, a poly A additionsignal, a selection marker, SV40 replication origin (hereinaftersometimes abbreviated as SV40ori) etc. Examples of the selection markerinclude dihydrofolate reductase (hereinafter sometimes abbreviated asdhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene(hereinafter sometimes abbreviated as Amp^(r)), neomycin resistant gene(hereinafter sometimes abbreviated as Neo^(r), G418 resistance), etc. Inparticular, when dhfr gene is employed as the selection marker usingdhfr gene-deficient Chinese hamster cells, selection can also be made onthymidine free media.

If necessary, a signal sequence that matches with a host is added to theN-terminus of the peptide of the present invention. Examples of thesignal sequence that can be used are Pho A signal sequence, OmpA signalsequence, etc. in the case of using bacteria of the genus Escherichia asthe host; α-amylase signal sequence, subtilisin signal sequence, etc. inthe case of using bacteria of the genus Bacillus as the host; MFα signalsequence, SUC2 signal sequence, etc. in the case of using yeast as thehost; and insulin signal sequence, α-interferon signal sequence,antibody molecule signal sequence, etc. in the case of using animalcells as the host, respectively.

Using the vector comprising the DNA encoding the peptide of the presentinvention thus constructed, transformants can be manufactured.

Examples of the host, which may be employed, are bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeast,insect cells, insects and animal cells, etc.

Specific examples of the bacteria belonging to the genus Escherichiainclude Escherichia coli K12 DH1 [Proc. Natl. Acad. Sci. U.S.A., 60, 160(1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journalof Molecular Biology, 120, 517 (1978)], HB101 [Journal of MolecularBiology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc.

Examples of the bacteria belonging to the genus Bacillus includeBacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal ofBiochemistry, 95, 87 (1984)], etc.

Examples of yeast include Saccharomyces cereviseae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036,Pichia pastoris KM71, etc.

Examples of insect cells include, for the virus AcNPV, Spodopterafrugiperda cell (Sf cell), MG1 cell derived from mid-intestine ofTrichoplusia ni, High Five™ cell derived from egg of Trichoplusia ni,cells derived from Mamestra brassicae, cells derived from Estigmenaacrea, etc.; and for the virus BmNPV, Bombyx mori N cell (BmN cell),etc. is used. Examples of the Sf cell which can be used are Sf9 cell(ATCC CRL1711), Sf21 cell (both cells are described in Vaughn, J. L. etal., In Vivo, 13, 213-217 (1977)), etc.

As the insect, for example, a larva of Bombyx mori, etc. can be used[Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinesehamster cell CHO (hereinafter referred to as CHO cell), dhfr genedeficient Chinese hamster cell CHO (hereinafter simply referred to asCHO (dhfr⁻) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, ratGH 3, human FL cell, etc.

Bacteria belonging to the genus Escherichia can be transformed, forexample, by the method described in Proc. Natl. Acad. Sci. U.S.A., 69,2110 (1972), Gene, 17, 107 (1982), etc.

Bacteria belonging to the genus Bacillus can be transformed, forexample, by the method described in Molecular & General Genetics, 168,111 (1979), etc.

Yeast can be transformed, for example, by the method described inMethods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci.U.S.A., 75, 1929 (1978), etc.

Insect cells or insects can be transformed, for example, according tothe method described in Bio/Technology, 6, 47-55(1988), etc.

Animal cells can be transformed, for example, according to the methoddescribed in Saibo Kogaku (Cell Engineering), extra issue 8, Shin SaiboKogaku Jikken Protocol (New Cell Engineering Experimental Protocol),263-267 (1995), published by Shujunsha, or Virology, 52, 456 (1973).

Thus, the transformant transformed with the expression vector containingthe DNA encoding the peptide can be obtained.

Where the host is bacteria belonging to the genus Escherichia or thegenus Bacillus, the transformant can be appropriately cultured in aliquid medium which contains materials required for growth of thetransformant such as carbon sources, nitrogen sources, inorganicmaterials, etc. Examples of the carbon sources include glucose, dextrin,soluble starch, sucrose, etc. Examples of the nitrogen sources includeinorganic or organic materials such as ammonium salts, nitrate salts,corn steep liquor, peptone, casein, meat extract, soybean cake, potatoextract, etc. Examples of the inorganic materials are calcium chloride,sodium dihydrogenphosphate, magnesium chloride, etc. In addition, yeast,vitamins, growth promoting factors etc. may also be added to the medium.Preferably, pH of the medium is adjusted to about 5 to about 8.

A preferred example of the medium for culturing the bacteria belongingto the genus Escherichia is M9 medium supplemented with glucose andCasamino acids [Miller, Journal of Experiments in Molecular Genetics,431-433, Cold Spring Harbor Laboratory, New York, 1972]. If necessaryand desired, a chemical such as 3β-indolylacrylic acid can be added tothe medium thereby to activate the promoter efficiently.

Where the bacteria belonging to the genus Escherichia are used as thehost, the transformant is usually cultivated at approximately 15 to 43°C. for approximately 3 to 24 hours. If necessary, the culture may beaerated or agitated.

Where the bacteria belonging to the genus Bacillus are used as the host,the transformant is cultivated generally at approximately 30 to 40° C.for approximately 6 to 24 hours. If necessary, the culture can beaerated or agitated.

Where yeast is used as the host, the transformant is cultivated, forexample, in Butkholder's minimal medium [Bostian, K. L. et al., Proc.Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in SD medium supplementedwith 0.5% Casamino acids [Bitter, G. A. et al., Proc. Natl. Acad. Sci.U.S.A., 81, 5330 (1984)]. Preferably, pH of the medium is adjusted toabout 5 to about 8. In general, the transformant is cultivated atapproximately 20° C. to 35° C. for approximately 24 to 72 hours. Ifnecessary, the culture can be aerated or agitated.

Where insect cells or insects are used as the host, the transformant iscultivated in, for example, Grace's Insect Medium (Grace, T. C. C.,Nature, 195, 788 (1962)) to which an appropriate additive such asimmobilized 10% bovine serum is added. Preferably, pH of the medium isadjusted to about 6.2 to about 6.4. Normally, the transformant iscultivated at about 27° C. for about 3 days to about 5 days and, ifnecessary, the culture can be aerated or agitated.

Where animal cells are employed as the host, the transformant iscultivated in, for example, MEM medium containing about 5% to about 20%fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], RPMI 1640 medium [The Journal of the American MedicalAssociation, 199, 519 (1967)], 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], etc. Preferably, pH of themedium is adjusted to about 6 to about 8. The transformant is usuallycultivated at about 30° C. to about 40° C. for about 15 hours to about60 hours and, if necessary, the culture can be aerated or agitated.

As described above, the peptide of the present invention or GPR7 can beproduced in the inside, cell membrane or outside of the transformant,etc.

The peptide of the present invention or GPR7 can be separated andpurified from the culture described above, e.g., by the followingprocedures.

When the peptide of the present invention or GPR7 is extracted from theculture or cells, the transformant or cell is collected, aftercultivation, by a publicly known method and suspended in an appropriatebuffer. The transformant or cell is then disrupted by publicly knownmethods such as ultrasonication, a treatment with lysozyme and/orfreeze-thaw cycling, followed by centrifugation, filtration, etc. Thus,the crude extract of the peptide of the present invention or GPR7 can beobtained. The buffer used for the procedures may contain a proteinmodifier such as urea or guanidine hydrochloride, or a surfactant suchas Triton X-100™, etc. When the peptide is secreted in the culturebroth, after completion of the cultivation the supernatant can beseparated from the transformant or cell to collect the supernatant by apublicly known method.

The peptide of the present invention or GPR7 contained in thesupernatant or in the extract thus obtained can be purified byappropriately combining the publicly known methods for separation andpurification. Such publicly known methods for separation andpurification include a method utilizing difference in solubility such assalting out, solvent precipitation, etc.; a method mainly utilizingdifference in molecular weight such as dialysis, ultrafiltration, gelfiltration, SDS-polyacrylamide gel electrophoresis, etc.; a methodutilizing difference in electric charge such as ion exchangechromatography, etc.; a method utilizing difference in specific affinitysuch as affinity chromatography, etc.; a method utilizing difference inhydrophobicity such as reversed phase high performance liquidchromatography, etc.; a method utilizing difference in isoelectric pointsuch as isoelectrofocusing electrophoresis; and the like.

When the peptide of the present invention or GPR7 thus obtained is in afree form, it can be converted into the salt by publicly known methodsor modifications thereof. On the other hand, when the peptide isobtained in the form of a salt, it can be converted into the free formor in the form of a different salt by publicly known methods ormodifications thereof.

The peptide of the present invention or GPR7 produced by the recombinantcan be treated, prior to or after the purification, with an appropriateprotein modifying enzyme so that the peptide can be appropriatelymodified to partially remove a peptide. Examples of theprotein-modifying enzyme include trypsin, chymotrypsin, arginylendopeptidase, protein kinase, glycosidase and the like.

Antibodies to the peptide of the present invention (hereinaftersometimes simply referred to as the antibody(ies) of the presentinvention) may be any of polyclonal antibodies and monoclonalantibodies, as long as they are capable of recognizing antibodies to thepeptide of the present invention.

The antibodies to the peptide of the present invention may bemanufactured by publicly known methods for manufacturing antibodies orantisera, using as antigens the peptide of the present invention.

[Production of Monoclonal Antibody]

(a) Production of Monoclonal Antibody-Producing Cells

The peptide of the present invention is administered to warm-bloodedanimals either solely or together with carriers or diluents to the sitewhere the production of antibody is possible by the administration. Inorder to potentiate the antibody productivity upon the administration,complete Freund's adjuvants or incomplete Freund's adjuvants may beadministered. The administration is usually carried out once every twoto six weeks and two to ten times in total. Examples of the applicablewarm-blooded animals are monkeys, rabbits, dogs, guinea pigs, rice,rats, sheep, goats and chickens, with the use of mice and rats beingpreferred.

In the preparation of monoclonal antibody-producing cells, awarm-blooded animal, e.g., mice, immunized with an antigen wherein theantibody titer is noted is selected, then spleen or lymph node iscollected after two to five days from the final immunization andantibody-producing cells contained therein are fused with myeloma cellsfrom homozoic or heterozoic animal to give monoclonal antibody-producinghybridomas. Measurement of the antibody titer in antisera may be carriedout, for example, by reacting a labeled peptide, which will be describedlater, with the antiserum followed by assaying the binding activity ofthe labeling agent bound to the antibody. The fusion may be carried out,for example, by the known method by Koehler and Milstein [Nature, 256,495 (1975)]. Examples of the fusion promoter are polyethylene glycol(PEG), Sendai virus, etc., of which PEG is preferably employed.

Examples of the myeloma cells are those collected from warm-bloodedanimals such as NS-1, P3U1, SP2/0, AP-1, etc. In particular, P3U1 ispreferably employed. A preferred ratio of the count of theantibody-producing cells used (spleen cells) to the count of myelomacells is within a range of approximately 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added in a concentration ofapproximately 10 to 80% followed by culturing at 20 to 40° C.,preferably at 30 to 37° C. for 1 to 10 minutes, an efficient cell fusioncan be carried out.

Various methods can be used for screening of a monoclonalantibody-producing hybridoma. Examples of such methods include a methodwhich comprises adding the supernatant of hybridoma to a solid phase(e.g., microplate) adsorbed with the peptide (protein) as an antigendirectly or together with a carrier, adding an anti-immunoglobulinantibody (where mouse cells are used for the cell fusion, anti-mouseimmunoglobulin antibody is used) labeled with a radioactive substance oran enzyme or Protein A and detecting the monoclonal antibody bound tothe solid phase, and a method which comprises adding the supernatant ofhybridoma to a solid phase adsorbed with an anti-immunoglobulin antibodyor Protein A, adding the peptide labeled with a radioactive substance oran enzyme and detecting the monoclonal antibody bound to the solidphase.

The monoclonal antibody can be selected according to publicly knownmethods or their modifications. In general, the selection can beeffected in a medium for animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). Any selection and growthmedium can be employed as far as the hybridoma can grow there. Forexample, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20%fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.)containing 1 to 10% fetal bovine serum, a serum free medium forcultivation of a hybridoma (SFM-101, Nissui Seiyaku Co., Ltd.) and thelike can be used for the selection and growth medium. The cultivation iscarried out generally at 20 to 40° C., preferably at 37° C., for about 5days to about 3 weeks, preferably 1 to 2 weeks, normally in 5% CO₂. Theantibody titer of the culture supernatant of a hybridoma can bedetermined as in the assay for the antibody titer in antisera describedabove.

(b) Purification of Monoclonal Antibody

Separation and purification of a monoclonal antibody can be carried outby publicly known methods, such as separation and purification ofimmunoglobulins [for example, salting-out, alcohol precipitation,isoelectric point precipitation, electrophoresis, adsorption anddesorption with ion exchangers (e.g., DEAE), ultracentrifugation, gelfiltration, or a specific purification method which comprises collectingonly an antibody with an activated adsorbent such as an antigen-bindingsolid phase, Protein A or Protein G and dissociating the binding toobtain the antibody].

[Production of Polyclonal Antibody]

The polyclonal antibody of the present invention can be manufactured bypublicly known methods or modifications thereof. For example, awarm-blooded animal is immunized with an immunogen (peptide antigen) perse, or a complex of immunogen and a carrier protein is formed and awarm-blooded animal is immunized with the complex in a manner similar tothe method described above for the manufacture of monoclonal antibodies.The product containing the antibody to the peptide of the presentinvention is collected from the immunized animal followed by separationand purification of the antibody.

In the complex of immunogen and carrier protein used to immunize awarm-blooded animal, the type of carrier protein and the mixing ratio ofcarrier to hapten may be any type and in any ratio, as long as theantibody is efficiently produced to the hapten immunized by crosslinkingto the carrier. For example, bovine serum albumin, bovine thyroglobulin,hemocyanin or the like is coupled to hapten in a carrier-to-haptenweight ratio of approximately 0.1 to 20, preferably about 1 to about 5.

A variety of condensation agents can be used for the coupling of carrierto hapten. Glutaraldehyde, carbodiimide, maleimide activated ester andactivated ester reagents containing thiol group or dithiopyridyl groupare used for the coupling.

The condensation product is administered to warm-blooded animals eithersolely or together with carriers or diluents to the site that canproduce the antibody by the administration. In order to potentiate theantibody productivity upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration is usually made once approximately every 2 to 6 weeks andapproximately 3 to 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc.,preferably from the blood of warm-blooded animal immunized by the methoddescribed above.

The polyclonal antibody titer in antiserum can be assayed by the sameprocedure as that for the determination of serum antibody titerdescribed above. The separation and purification of the polyclonalantibody can be carried out, following the method for the separation andpurification of immunoglobulins performed as in the separation andpurification of monoclonal antibodies described hereinabove.

Antisense DNAs (hereinafter these DNAs are sometimes merely referred toas the antisense DNA) having a complementary or substantiallycomplementary base sequence to the DNA encoding the peptide of thepresent invention (hereinafter these DNAs are sometimes merely referredto as the DNA of the present invention) can be any antisense DNA, solong as they possess a base sequence complementary or substantiallycomplementary to that of the DNA of the present invention and capable ofsuppressing expression of the DNA.

The base sequence substantially complementary to the DNA of the presentinvention may, for example, be a base sequence having at least about 70%homology, preferably at least about 80% homology, more preferably atleast about 90% homology and most preferably at least about 95%homology, to the full-length base sequence or partial base sequence ofthe base sequence complementary to the DNA of the present invention(i.e., complementary strand to the DNA of the present invention). In theentire base sequence of the complementary strand to the DNA of thepresent invention, an antisense DNA having at least about 70% homology,preferably at least about 80% homology, more preferably at least about90% homology and most preferably at least about 95% homology, to thecomplementary strand of the base sequence which encodes the N-terminalregion of the peptide of the present invention (e.g., the base sequencearound the initiation codon). These antisense DNAs can be synthesizedusing a publicly known DNA synthesizer, etc.

Hereinafter, the utilities of (1) the peptide of the present invention;(2) the DNA of the present invention, (3) the antibody of the presentinvention, and (4) the antisense DNA are explained.

(1) Therapeutic/Preventive Agent for Diseases with which the Peptide ofthe Present Invention is Associated

As shown in EXAMPLE 6 later described, the peptide of the presentinvention has the cell stimulating activity on GPR7-expressed cells(e.g., the activity that promotes arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, GTPγ Sbinding activity, etc.), and is an endogenous ligand to GPR7. Moreover,the peptide of the present invention has an appetite (eating)stimulating activity, as shown in EXAMPLE 14 later described. Besides,the peptide of the present invention is considered to act as aneuromodulator or neuroendocrine substance or to be associated withmemory, learning or stress control.

Therefore, when the peptide of the present invention or the DNA of thepresent invention involves any abnormality or deficiency, or when GPR7or the DNA encoding GPR7 involves any abnormality or deficiency, it ishighly likely to cause various diseases, including anorexia,hypertension, autoimmune disease, heart failure, cataract, glaucoma,acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, nbn-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative diseases (especially anorexia, etc.), or the like.

Therefore, the peptide of the present invention and the DNA of thepresent invention can be used as pharmaceuticals (in particular,appetite (eating) stimulants, etc.) for the treatment/prevention ofvarious diseases as described above (especially anorexia, etc.).

When a patient has a reduced level of, or deficient in the peptide ofthe present invention in his or her body, the peptide of the presentinvention and the DNA of the present invention can provide the role ofthe peptide of the present invention sufficiently or properly for thepatient, (a) by administering the DNA of the present invention to thepatient to express the peptide of the present invention in the body, (b)by inserting the DNA of the present invention into a cell, expressingthe peptide of the present invention and then transplanting the cell tothe patient, or (c) by administering the peptide of the presentinvention to the patient, or the like.

When the DNA of the present invention is used as thepreventive/therapeutic agents described above, the DNA is administereddirectly to human or other warm-blooded animal; alternatively, the DNAis inserted into an appropriate vector such as retrovirus vector,adenovirus vector, adenovirus-associated virus vector, etc. and thenadministered to human or other warm-blooded animal in a conventionalmanner. The DNA of the present invention may also be administered as anintact DNA, or prepared into pharmaceutical preparations together with aphysiologically acceptable carrier such as an adjuvant to assist itsuptake and administered by gene gun or through a catheter such as acatheter with a hydrogel.

Where the peptide of the present invention is used as the aforesaidtherapeutic/preventive agents, the peptide is advantageously used on apurity level of at least 90%, preferably at least 95%, more preferablyat least 98% and most preferably at least 99%.

The peptide of the present invention can be used orally, for example, inthe form of tablets which may be sugar coated if necessary, capsules,elixirs, microcapsules etc., or parenterally in the form of injectablepreparations such as a sterile solution and a suspension in water orwith other pharmaceutically acceptable liquid. These preparations can bemanufactured by mixing the peptide of the present invention with aphysiologically acceptable known carrier, a flavoring agent, anexcipient, a vehicle, an antiseptic agent, a stabilizer, a binder, etc.in a unit dosage form required in a generally accepted manner that isapplied to making pharmaceutical preparations. The active ingredient inthe preparation is controlled in such a dose that an appropriate dose isobtained within the specified range given.

Additives miscible with tablets, capsules, etc. include a binder such asgelatin, corn starch, tragacanth and gum arabic, an excipient such ascrystalline cellulose, a swelling agent such as corn starch, gelatin,alginic acid, etc., a lubricant such as magnesium stearate, a sweeteningagent such as sucrose, lactose and saccharin, and a flavoring agent suchas peppermint, akamono oil or cherry, etc. When the unit dosage is inthe form of capsules, liquid carriers such as oils and fats may furtherbe used together with the additives described above. A sterilecomposition for injection may be formulated according to a conventionalmanner used to make pharmaceutical compositions, e.g., by dissolving orsuspending the active ingredients in a vehicle such as water forinjection with a naturally occurring vegetable oil such as sesame oiland coconut oil, etc. to prepare the pharmaceutical composition.

Examples of an aqueous medium for injection include physiological salineand an isotonic solution containing glucose and other auxiliary agents(e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be used incombination with an appropriate dissolution aid such as an alcohol(e.g., ethanol or the like), a polyalcohol (e.g., propylene glycol andpolyethylene glycol), a nonionic surfactant (e.g., polysorbate 80™ andHCO-50), etc. Examples of the oily medium include sesame oil and soybeanoil, which may also be used in combination with a dissolution aid suchas benzyl benzoate and benzyl alcohol. The prophylactic/therapeuticagent described above may further be formulated with a buffer (e.g.,phosphate buffer, sodium acetate buffer, etc.), a soothing agent (e.g.,benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer(e.g., human serum albumin, polyethylene glycol, etc.), a preservative(e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. Thethus-prepared liquid for injection is normally filled in an appropriateampoule.

The vector in which the DNA of the present invention is inserted mayalso be prepared into pharmaceutical preparations in a manner similar tothe procedures above. Such preparations are generally used parenterally.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation can be administered to human or otherwarm-blooded animals (e.g., rats, mice, guinea pigs, rabbits, chicken,sheep, swine, bovine, horses, cats, dogs, monkeys, etc.).

The dose of the peptide of the present invention varies depending ontarget disease, subject to be administered, route for administration,etc.; in oral administration, e.g., for the treatment of anorexia, thedose is normally about 0.1 mg to about 100 mg, preferably about 1.0 toabout 50 mg, and more preferably about 1.0 to about 20 mg per day foradult (as 60 kg body weight). In parenteral administration, the singledose varies depending on subject to be administered, target disease,etc. but it is advantageous, e.g., for the treatment of anorexia toadminister the active ingredient intravenously at a daily dose of about0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and morepreferably about 0.1 to about 10 mg for adult (as 60 kg body weight).For other animal species, the corresponding dose as converted per 60 kgbody weight can be administered.

(2) Screening of Drug Candidate Compounds for Diseases

(2-1) Screening Method A

Since the peptide of the present invention has the function to act asthe ligand to GPR7, the compounds or salts thereof that promote thefunction of the peptide of the present invention can be used as drugsfor the treatment/prevention of diseases such as anorexia, hypertension,autoimmune disease, heart failure, cataract, glaucoma, acute bacterialmeningitis, acute myocardial infarction, acute pancreatitis, acute viralencephalitis, adult respiratory distress syndrome, alcoholic hepatitis,Alzheimer's disease, asthma, arteriosclerosis, atopic dermatitis,bacterial pneumonia, bladder cancer, fracture, breast cancer, bulimia,polyphagia, burn healing, uterine cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic pancreatitis, livercirrhosis, cancer of the colon and rectum (colon cancer/rectal cancer),Crohn's disease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia, etc.), or the like.

On the other hand, the compounds or salts thereof that inhibit thefunction of the peptide of the present invention are useful as safe andlow-toxic drugs for the prevention/treatment of, e.g., obesity (e.g.,malignant mastocytosis, exogenous obesity, hyperinsulinar obesity,hyperplasmic obesity, hypophyseal adiposity, hypoplasmic obesity,hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, etc.; as safe and low-toxic drugs for thetreatment/prevention (prolactin production suppressing agents) forpituitary tumor, diencephalon tumor, menstrual disorder, autoimmunedisease, prolactinoma, sterility, impotence, amenorrhea, lactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc., preferably, as safe and low-toxic drugsfor the prevention/treatment of obesity, hyperphagia, etc.

By using the peptide of the present invention, or by constructing theexpression system of the recombinant peptide of the present inventionand using the receptor-binding assay system via the expression system,compounds that alter the binding property between the peptide of thepresent invention and GPR7 (compounds that promote or inhibit theactivities of the peptide of the present invention) (e.g., peptide,protein, a non-peptide compound, a synthetic compound, fermentationproduct, etc.), or salts thereof, can be screened. Such compoundsinclude compounds (i.e., GPR7 agonists) that have the cell-stimulatingactivity of the peptide of the present invention (e.g., the activitythat promotes arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.) mediated by GPR7;compounds having no such cell-stimulating activity (i.e., GPR7antagonists); and the like. The term “alters the binding property to theligand” is used to include both cases where binding to the ligand isinhibited and binding to the ligand is promoted.

Thus, the present invention provides:

a method of screening a compound or its salt that promotes or inhibitsthe activity of the peptide of the present invention, which comprisesusing the peptide of the present invention, more specifically:

a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises comparing (i) the case whereinthe peptide of the present invention is brought in contact with GPR7 orits partial peptide (hereinafter they are sometimes merely referred toas GPR7) and (ii) the case wherein the peptide of the present inventionand a test compound are brought in contact with GPR7.

According to the screening method of the present invention, the methodcomprises assaying, for example, the binding amount of the ligand toGPR7, the cell-stimulating activity, etc. (i) in the case wherein thepeptide of the present invention is brought in contact with GPR7 and(ii) in the case wherein the peptide of the present invention and a testcompound are brought in contact with GPR7, and comparing (i) and (ii).

Specifically, the screening method of the present invention includes:

(1) a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises assaying the binding amount of alabeled form of the peptide of the present invention to GPR7, (i) in thecase wherein a labeled form of the peptide of the present invention isbrought in contact with GPR7 and (ii) in the case wherein a labeled formof the peptide of the present invention and a test compound are broughtin contact with GPR7, and comparing (i) and (ii);

(2) a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises assaying the binding amount of alabeled form of the peptide of the present invention to a cellcontaining GPR7 or its cell membrane, (i) in the case wherein a labeledform of the peptide of the present invention is brought in contact withthe cell containing GPR7 or its cell membrane and (ii) in the casewherein a labeled form of the peptide of the present invention and atest compound are brought in contact with the cell containing GPR7 orits cell membrane, and comparing (i) and (ii);

(3) a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises assaying the binding amount of alabeled form of the peptide of the present invention to GPR7, (i) in thecase wherein a labeled form of the peptide of the present invention isbrought in contact with GPR7 expressed on a cell membrane by culturing atransformant containing a DNA encoding GPR7 and (ii) in the case whereina labeled form of the peptide of the present invention and a testcompound are brought in contact with GPR7 expressed on a cell membraneby culturing a transformant containing a DNA encoding GPR7, andcomparing (i) and (ii);

(4) a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises assaying the cell-stimulatingactivity mediated by GPR7 (e.g., the activity that promotes orsuppresses arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.), when a compoundthat activates GPR7 (e.g., the peptide of the present invention) isbrought in contact with a cell containing GPR7 and when the compoundthat activates GPR7 and a test compound are brought in contact with acell containing GPR7, and comparing the activity; and,

(5) a method of screening a compound that alters the binding propertybetween the peptide of the present invention and GPR7 (a compound thatpromotes or inhibits the activity of the peptide of the presentinvention) or its salt, which comprises assaying the cell-stimulatingactivity mediated by GPR7 (e.g., the activity that promotes orsuppresses arachidonic acid release, acetylcholine release,intracellular Ca²⁺ release, intracellular cAMP production, intracellularcGMP production, inositol phosphate production, change in cell membranepotential, phosphorylation of intracellular proteins, activation ofc-fos, pH reduction, GTPγ S binding activity, etc.), when a compoundthat activates GPR7 (e.g., the peptide of the present invention, etc.)is brought in contact with GPR7 expressed on a cell membrane byculturing a transformant containing a DNA encoding GPR7 and when thecompound that activates GPR7 and a test compound are brought in contactwith GPR7 expressed on a cell membrane by culturing a transformantcontaining a DNA encoding GPR7, and comparing the activity; etc.

The screening method of the present invention will be described belowmore specifically.

First, the GPR7, which is used for the screening method of the presentinvention, may be any protein, so long as it recognizes the peptide ofthe present invention as a ligand, and membrane fractions from human orother warm-blooded animal organs are preferably employed. However, it isvery difficult to obtain human-derived organs especially, and the GPR7,etc. expressed abundantly by use of recombinants are suitable for use inthe screening. GPR7 may be manufactured by the methods described above.

Where the cell containing GPR7 or its cell membrane fraction is used inthe screening method of the present invention, the procedures laterdescribed may apply.

When the cell containing GPR7 is used, the cell may be fixed withglutaraldehyde, formalin, etc. The fixation may be carried out by apublicly known method.

The cell containing GPR7 refers to a host cell expressing GPR7. Examplesof such a host cell include Escherichia coli, Bacillus subtilis, yeast,insect cells, animal cells, etc. Host cells in which GPR7 is expressedmay be prepared in a manner similar to the above-stated method formanufacturing transformants transformed by expression vectors containingthe peptide of the present invention.

The membrane fraction refers to a fraction that abundantly contains cellmembranes prepared by publicly known methods after disrupting cells.Examples of the cell disruption include cell squashing using aPotter-Elvehjem homogenizer, disruption using a Waring blender orPolytron (manufactured by Kinematica Inc.), disruption byultrasonication, disruption by cell spraying via a thin nozzle underincreasing pressure using a French press, etc., and the like. Cellmembranes are fractionated mainly by fractionation using a centrifugalforce such as for fractionation centrifugation, density gradientcentrifugation, etc. For example, cell disruption fluid is centrifugedat a low rate (500 rpm to 3,000 rpm) for a short period of time(normally about 1 minute to about 10 minutes), the resulting supernatantis then centrifuged at a higher rate (15,000 rpm to 30,000 rpm) normallyfor 30 minutes to 2 hours. The precipitate thus obtained is used as themembrane fraction. The membrane fraction is rich in GPR7 expressed andmembrane components such as cell-derived phospholipids, membraneproteins, or the like.

The amount of GPR7 contained in the cells containing GPR7 or in themembrane fraction is preferably 10³ to 10⁸ molecules per cell, morepreferably 10⁵ to 10⁷ molecules per cell. As the amount of expressionincreases, the ligand binding activity per unit of membrane fraction(specific activity) increases so that not only the highly sensitivescreening system can be constructed but also large quantities of samplescan be assayed with the same lot.

To perform the methods (1) through (3) for screening the compound thatalters the binding property between the peptide of the present inventionand GPR7 (the compound that promotes or inhibits the activity of thepeptide of the present invention), an appropriate GPR7 fraction and alabeled form of the peptide of the present invention, etc. are required.The GPR7 fraction is preferably a fraction of a naturally occurring formof GPR7 or a fraction of a recombinant type of GPR7 having an equivalentactivity. Herein, the term equivalent activity is intended to mean aligand binding activity, etc. that is equivalent to the activitypossessed by naturally occurring GPR7. As the labeled ligand, there maybe used a labeled ligand, a labeled ligand analog compound, etc. Forexample, there may be used ligands that are labeled with [³H], [¹²⁵I],[¹⁴C], [³⁵S], etc. Of these, [¹²⁵I]-labeled ligand is preferred.

Specifically, the compound that alters the binding property between thepeptide of the present invention and GPR7 is screened by the followingprocedures. First, a receptor preparation is prepared by suspendingcells containing GPR7 or the membrane fraction thereof in a bufferappropriate for use in the screening method. Any buffer can be used solong as it does not interfere the ligand-receptor binding, including aphosphate buffer or a Tris-HCl buffer, having pH of 4 to 10 (preferablypH of 6 to 8), etc. For the purpose of minimizing non-specific binding,a surfactant such as CHAPS, Tween-80™ (Kao-Atlas Inc.), digitonin,deoxycholate, etc., may optionally be added to the buffer. Further forthe purpose of suppressing the degradation of GPR7 or the peptide of thepresent invention with a protease, a protease inhibitor such as PMSF,leupeptin, E-64 (manufactured by Peptide Institute, Inc.), pepstatin,etc. may also be added. A given amount (5,000 cpr to 500,000 cpm) of thelabeled peptide of the present invention is added to 0.01 ml to 10 ml ofthe receptor solution, in which 10⁻¹⁰ M to 10⁻⁷ M of a test compound isco-present. To determine the amount of non-specific binding (NSB), areaction tube charged with an unlabeled form of the peptide of thepresent invention in a large excess is also provided. The reaction iscarried out at approximately 0° C. to 50° C., preferably 4° C. to 37° C.for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. Aftercompletion of the reaction, the reaction mixture is filtrated throughglass fiber filter paper, etc. and washed with an appropriate volume ofthe same buffer. The residual radioactivity on the glass fiber filterpaper is then measured by means of a liquid scintillation counter orγ-counter. When nonspecific binding (NSB) is subtracted from the count(B₀) where any antagonizing substance is absent and the resulting count(B₀ minus NSB) is made 100%, the test compound showing the specificbinding amount (B minus NSB) of, e.g., 50% or less may be selected as acandidate compound.

The method (4) or (5) described above for screening the compound thatalters the binding property between the peptide of the present inventionand GPR7 (the compound that promotes or inhibits the activity of thepeptide of the present invention) can be carried out as follows. Forexample, the cell stimulating activity mediated by GPR7 (e.g., theactivity that promotes or suppresses arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, change in cell membrane potential, phosphorylation ofintracellular proteins, activation of c-fos, pH reduction, GTPγ Sbinding activity, etc.) may be determined by a publicly known method, orusing an assay kit commercially available. Specifically, the cellscontaining GPR7 are first cultured on a multiwell plate, etc. Prior toscreening, the medium is replaced with fresh medium or with anappropriate non-cytotoxic buffer, followed by incubation for a givenperiod of time in the presence of a test compound, etc. Subsequently,the cells are extracted or the supernatant is recovered and theresulting product is quantified by appropriate procedures. Where it isdifficult to detect the production of the cell-stimulating activityindicator (e.g., arachidonic acid, etc.) due to a degrading enzymecontained in the cells, an inhibitor against such as a degrading enzymemay be added prior to the assay. For detecting the activity such as thecAMP production suppression, the baseline production in the cells isincreased by forskolin or the like and the suppressing effect on theincreased baseline production can be detected.

For screening through the assay of the cell stimulating activity,appropriate cells, in which GPR7 is expressed, are required. Preferredcells, in which GPR7 is expressed, are the aforesaid cell line in whichGPR7 is expressed, etc.

Examples of the test compounds include peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,plant extracts, animal tissue extracts, etc.

The kit for screening the compound or a salt thereof that alters thebinding property between the peptide of the present invention (thecompound that promotes or inhibits the activity of the peptide of thepresent invention) and GPR7 comprises GPR7 or its salt, a partialpeptide of GPR7 or its salt, cells containing GPR7 or a membranefraction of the cells containing GPR7, and the peptide of the presentinvention.

Examples of the screening kit of the present invention are given below:

1. Reagent for Screening

(1) Assay Buffer and Wash Buffer

Hanks' Balanced Salt Solution (manufactured by Gibco Co.) supplementedwith 0.05% bovine serum albumin (Sigma Co.).

The solution is sterilized by filtration through a 0.45 μm filter andstored at 4° C. Alternatively, the solution may be prepared at use.

(2) GPR7 Preparation

CHO cells on which GPR7 has been expressed are subcultured in a 12-wellplate at the rate of 5×10⁵ cells/well and then cultured at 37° C. under5% CO₂ and 95% air for 2 days.

(3) Labeled Ligand

The peptide of the present invention labeled with commercially available[³H], [¹²⁵I], [¹⁴C], [³⁵S], etc. is dissolved in a suitable solvent orbuffer. The solution is stored at 4° C. or −20° C., which is diluted to1 μM with an assay buffer at use.

(4) Standard Ligand Solution

The peptide of the present invention is dissolved in PBS supplementedwith 0.1% bovine serum albumin (manufactured by Sigma, Inc.) in aconcentration of 1 mM, and the solution is stored at −20° C.

2. Assay Method

(1) Cells are cultured in a 12-well tissue culture plate to expressGPR7. After washing the cells twice with 1 ml of the assay buffer, 490μl of the assay buffer is added to each well.

(2) After 5 μl of a test compound solution of 10⁻³ to 10⁻¹⁰ M is added,5 μl of a labeled form of the peptide of the present invention is addedto the system followed by reacting at room temperature for an hour. Todetermine the amount of the non-specific binding, the peptide of thepresent invention of 10⁻³ M is added in an amount of 5 μl, instead ofthe test compound.

(3) The reaction mixture is removed and washed 3 times with 1 ml each ofthe wash buffer. The labeled peptide of the present invention bound tothe cells is dissolved in 0.2N NaOH-1% SDS and mixed with 4 ml of aliquid scintillator A (manufactured by Wako Pure Chemical Industries,Ltd.).

(4) Radioactivity is measured using a liquid scintillation counter(manufactured by Beckmann) and PMB (percent of the maximum binding) iscalculated in accordance with the following equation 1:PMB=[(B−NSB)/(B ₀ −NSB)]×100wherein:

PMB: percent of the maximum binding

B: value when a sample is added

NSB: non-specific binding

B₀: maximum binding

The compound or its salt, which can be obtained by the screening methodor the screening kit of the present invention, is the compound thatalters the binding property between the peptide of the present inventionand GPR7 (the compound that promotes or inhibits the activity of thepeptide of the present invention). Specifically, these compounds arecompounds or salts thereof that exhibit the cell stimulating activitymediated by GPR7 (i.e., GPR7 agonist), or compounds that have no suchcell stimulating activity (i.e., GPR7 antagonist). Examples of suchcompounds-include peptides, proteins, non-peptide compounds, syntheticcompounds and fermentation products. These compounds may be either novelor publicly known compounds.

In order to evaluate whether the compound is either the GPR7 agonist orantagonist described above, it is determined by (i) or (ii) below.

(i) According to the screening methods (1) to (3), binding assay iscarried out to obtain the compound that alters the binding propertybetween the peptide of the present invention and GPR7 (especially, thecompound that inhibits the binding). It is then determined if thecompound has the above cell-stimulating activity mediated by GPR7. Thecompound or its salt having the cell-stimulating activity is the GPR7agonist, whereas the compound or its salt having no such an activity isthe GPR7 antagonist.

(ii) (a) A test compound is brought in contact with a cell containingGPR7, whereby the aforesaid cell-stimulating activity mediated by GPR7is assayed. The compound having the cell-stimulating activity or itssalt is the GPR7 agonist.

(b) The cell-stimulating activity mediated by GPR7 is assayed in thecase where a compound that activates GPR7 (e.g., the peptide of thepresent invention or GPR7 agonist, etc.) is brought in contact withcells containing GPR7 and in the case where the compound that activatesGPR7 and a test compound are brought in contact with cells containingGPR7, and comparison is made therebetween. The compound or its salt thatcan reduce the cell-stimulating activity induced by the compound thatactivates GPR7 is the GPR7 antagonist.

The GPR7 agonists exhibit similar physiological activity of the peptideof the present invention on GPR7, and are thus safe and low-toxic drugs(e.g., preventive/therapeutic drugs for anorexia, appetite (eating)stimulants, preventive/therapeutic drugs for pituitary hormone secretiondisorders [e.g., prolactin secretion disorders (e.g., hypoovarianism,spermatic underdevelopment, menopausal symptoms, hypothyroidism, etc.)].

On the contrary, the GPR7 antagonist can suppress the physiologicalactivity that the peptide of the present invention has on GPR7, and arethus useful as safe and low-toxic drugs for the prevention/treatment of,e.g., obesity (e.g., malignant mastocytosis, exogenous obesity,hyperinsulinar obesity, hyperplasmic obesity, hypophyseal adiposity,hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity,symptomatic obesity, infantile obesity, upper body obesity, alimentaryobesity, hypogonadal obesity, systemic mastocytosis, simple obesity,central obesity, etc.), hyperphagia, etc.; as safe and low-toxic drugsfor the treatment/prevention (prolactin production suppressing agents)for pituitary tumor, diencephalon tumor, menstrual disorder, autoimmunedisease, prolactinoma, sterility, impotence, amenorrhea, lactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc.; preferably as safe and low-toxicpreventive/therapeutic agents for obesity, hyperphagia, etc.

The compound or its salt, which can be obtained by using the screeningmethod A or the screening kit of the present invention, is selectedfrom, e.g., peptides, proteins, non-peptide compounds, syntheticcompounds, fermentation products, cell extracts, plant extracts, animaltissue extracts, plasma, etc., and is the compound that promotes orinhibits the function of the peptide of the present invention.

As salts of the compound, those similar to the salts of the peptide ofthe present invention described above may be used.

When the compound obtained by the screening method A or screening kit ofthe present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations in a conventional manner. For example, the compound may beprepared in the form of tablets, capsules, elixir, microcapsule, asterile solution, a suspension, etc., as in the aforesaid drugscontaining the peptide of the present invention.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation may be administered to human or otherwarm-blooded animals (e.g., mice, rats, rabbits, sheep, swine, bovine,horses, chicken, cats, dogs, monkeys, chimpanzees, etc.).

The dose of the compound or its salt varies depending on its activity,target disease, subject to be administered, route for administration,etc.; where the GPR7 agonist is orally administered, e.g., for thetreatment of anorexia, the dose of the compound is normally about 0.1 toabout 100 mg, preferably about 1.0 to about 50 mg, and more preferablyabout 1.0 to about 20 mg per day for adult (as 60 kg body weight). Inparenteral administration, a single dose of the compound variesdepending on subject to be administered, target disease, etc. When theGPR7 agonist is administered to adult (as 60 kg body weight) in the formof injection, e.g., for the treatment of anorexia, it is advantageous toadminister the compound intravenously to adult generally at a daily doseof about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, andmore preferably about 0.1 to about 10 mg. For other animal species, thecorresponding dose as converted per 60 kg body weight can beadministered.

Also, when the GPR7 antagonist is orally administered to adult (per 60kg body weight), e.g., for the treatment of obesity, a daily dose of thecompound administered is generally approximately 0.1 to 100 mg,preferably approximately 1.0 to 50 mg, and more preferably approximately1.0 to 20 mg. In parenteral administration, a single dose of thecompound varies depending on subject to be administered, target disease,etc. When the GPR7 antagonist is administered to adult (as 60 kg bodyweight) in the form of injection, e.g., for the treatment of obesity, itis advantageous to administer the compound intravenously to adult (per60 kg body weight) generally at a daily dose of about 0.01 to about 30mg, preferably about 0.1 to about 20 mg, and more preferably about 0.1to about 10 mg. For other animal species, the corresponding dose asconverted per 60 kg body weight can be administered.

(2-2) Screening Method B

Next, the method of screening a compound that regulates the expressionlevel of GPR7 ligand is explained below.

The screening method B of the present invention is specifically (i) amethod of screening a compound or its salt that increases or decreasesthe expression level of GPR7 ligand, which comprises assaying theexpression level of GPR7 ligand or the amount of mRNA encoding GPR7ligand in the case that a cell or tissue capable of expressing GPR7ligand is cultured in the presence or absence of a test compound, andcomparing the expression level in each case.

As the cell or tissue capable of expressing GPR7 ligand, there may beused a cell or tissue derived from human or other warm-blooded animals(e.g. guinea pigs, rats, mice, chicken, rabbits, swine, sheep, bovine,monkeys, etc.); any cell (e.g., nerve cells, endocrine cells,neuroendocrine cells, glial cells, β cells of pancreas, bone marrowcells, hepatocytes, splenocytes, mesangial cells, epidermic cells,epithelial cells, endothelial cells, fibroblasts, fibrocytes, myocytes,fat cells, immune cells (e.g., macrophages, T cells, B cells, naturalkiller cells, mast cells, neutrophils, basophils, eosinophils,monocytes, dendritic cells), megakaryocyte, synovial cells,chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cellsor interstitial cells, the corresponding precursor cells, stem cells,cancer cells, etc., of these cells), or any tissue where such a cell ispresent, e.g., brain or any region of the brain (e.g., olfactory bulb,amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus,cerebral cortex, medulla oblongata, cerebellum), spinal cord,hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, testis, ovary, placenta, uterus, bone, cartilage,joint, skeletal muscle, etc., wherein established cell line or primaryculture system may also be used. Transformants transformed by arecombinant vector bearing a DNA encoding GPR7 ligand described abovemay also be used.

To cultivate the cells capable of expressing GPR7 ligand, the methodgiven for cultivating transformants above applies.

As the test compound, a DNA library may also be used, in addition to thetest compounds described above.

The expression level of GPR7 ligand can be determined by publicly knownmethods such as immunochemical methods, etc., using an antibody, etc.Alternatively, mRNA encoding GPR7 ligand can be determined by publiclyknown methods including northern hybridization, RT-PCR or TaqMan PCR.

Comparison of the expression level of mRNA can be made by publicly knownmethods or a modification thereof, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989).

Specifically, the amount of mRNA encoding GPR7 ligand is determined bycontacting RNA extracted from cells according to publicly known methodswith the DNA encoding GPR7 ligand or a part thereof or the antisensepolynucleotide of the present invention, and assaying the amount of mRNAbound to the DNA encoding GPR7 ligand or a part thereof or the antisensepolynucleotide of the present invention. The amount of mRNA bound to theDNA encoding GPR7 ligand or a part thereof or the antisensepolynucleotide of the present invention can be readily assayed bylabeling the DNA encoding GPR7 ligand or a part thereof or the antisensepolynucleotide of the present invention with, e.g., a radioisotope, adye, etc. Examples of the radioisotope are ³²P, ³H, etc. Examples of thedye used are fluorescent dyes such as fluorescein, FAM (Biosystems,Inc.), JOE (PE Biosystems, Inc.), TAMRA (PE Biosystems, Inc.), ROX (PEBiosystems, Inc.), Cy5 (Amersham), Cy3 (Amersham), etc.

The amount of mRNA can also be determined by converting RNA extractedfrom cells into cDNA by a reverse transcriptase, amplifying the cDNA byPCR using the DNA encoding GPR7 ligand or a part thereof or theantisense polynucleotide of the present invention as a primer, andassaying the amount of cDNA amplified.

As described above, the test compound that increases the amount of mRNAencoding GPR7 ligand can be selected as a compound that increases theexpression level of GPR7 ligand. Also, the test compound that decreasesthe amount of mRNA encoding GPR7 ligand can be selected as a compoundthat decreases the expression level of GPR7 ligand.

The present invention further provides:

(ii) a method of screening a compound or its salt that promotes orinhibits a reporter activity, which comprises assaying the expressionlevel of GPR7 ligand or the amount of mRNA encoding GPR7 ligand in thecase that a cell or tissue capable of expressing GPR7 ligand is culturedin the presence or absence of a test compound, and comparing theexpression level in each case.

As the reporter gene, there may be employed, e.g., lacZ (β-galactosidasegene), chloramphenicol acetyltransferase (CAT), luciferase, growthfactor, β-glcuronidase, alkaline phosphatase, green fluorescent protein(GFP), β-lactamase, etc.

By determining the level of the reporter gene product (e.g., mRNA,protein) Using publicly known methods, the test compound that increasesthe level of the reporter gene product can be selected as the compoundhaving the activity of regulating (especially promoting) the promoter orenhancer activity of GPR7 ligand of the present invention, i.e., thecompound having the activity of increasing the expression level of GPR7ligand. To the contrary, the test compound that decreases the level ofthe reporter gene product can be selected as the compound having theactivity of regulating (especially inhibiting) the promoter or enhanceractivity of GPR7 ligand, i.e., the compound having the activity ofdecreasing the expression level of GPR7 ligand.

As the test compounds, those described above are employed.

The transformants can be cultivated as in the transformants describedabove.

Construction of vectors for the reporter genes and assay can beperformed according to publicly known techniques (e.g., MolecularBiotechnology, 13, 29-43, 1999).

The compounds having the activity of increasing the expression level ofGPR7 are useful as safe and low-toxic drugs (e.g.,preventive/therapeutic agents for anorexia, appetite (eating)stimulants, preventive/therapeutic agents for pituitary hormonesecretion disorders [e.g., prolactin secretion disorders (e.g.,hypoovarianism, spermatic underdevelopment, menopausal symptoms,hypothyroidism, etc.)].

The compounds having the activity of decreasing the expression level ofGPR7 ligand are useful as safe and low-toxic drugs for theprevention/treatment of obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, etc.; as safe andlow-toxic drugs for the prevention/treatment (prolactin productionsuppressing agents) for pituitary tumor, diencephalon tumor, menstrualdisorders, autoimmune disease, prolactinoma, sterility, impotence,amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-delCastillo syndrome, Forbes-Albright syndrome, lymphoma or Sheehan'ssyndrome, spermatogenesis disorder, etc.; preferably, as safe andlow-toxic drugs for the prevention/treatment of obesity, hyperphagia,etc.

The compound or its salt, which can be obtained by using the screeningmethod B or the screening kit of the present invention, is a compoundselected from, e.g., peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts, plasma, etc., and is the compound thatpromotes or inhibits the function of the peptide of the presentinvention.

For salts of the compound, those as described for the peptide of thepresent invention are employed.

When the compound obtained by the screening method B or screening kit ofthe present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations in a conventional manner. For example, the compound may beprepared in the form of tablets, capsules, elixir, microcapsule, asterile solution, a suspension, etc., as in the aforesaid drugscontaining the peptide of the present invention.

Since the thus obtained pharmaceutical preparation is safe and lowtoxic, the preparation may be administered to human or otherwarm-blooded animals (e.g., mice, rats, rabbits, sheep, swine, bovine,horses, chicken, cats, dogs, monkeys, chimpanzees, etc.).

The dose of the compound or its salt varies depending on its activity,target disease, subject to be administered, route for administration,etc.; where the compound that increases the expression level of GPR7ligand is orally administered, e.g., for the treatment of anorexia, thedose of the compound is normally about 0.1 to about 100 mg, preferablyabout 1.0 to about 50 mg, and more preferably about 1.0 to about 20 mgper day for adult (as 60 kg body weight). In parenteral administration,a single dose of the compound varies depending on subject to beadministered, target disease, etc. When the compound that increases theexpression level of GPR7 ligand is administered to adult (as 60 kg bodyweight) in the form of injection, e.g., for the treatment of anorexia,it is advantageous to administer the compound intravenously to adultgenerally at a daily dose of about 0.01 to about 30 mg, preferably about0.1 to about 20 mg, and more preferably about 0.1 to about 10 mg. Forother animal species, the corresponding dose as converted per 60 kg bodyweight can be administered.

Also, when the compound that decreases the expression level of GPR7ligand is orally administered to adult (per 60 kg body weight), e.g.,for the treatment of obesity, a daily dose of the compound administeredis generally approximately 0.1 to 100 mg, preferably approximately 1.0to 50 mg, and more preferably approximately 1.0 to. 20 mg. In parenteraladministration, a single dose of the compound varies depending onsubject to be administered, target disease, etc. When the compound thatdecreases the expression level of GPR7 ligand is administered to adult(as 60 kg body weight) in the form of injection, e.g., for the treatmentof obesity, it is advantageous to administer the compound intravenouslyto adult (per 60 kg body weight) generally at a daily dose of about 0.01to about 30 mg, preferably about 0.1 to about 20 mg, and more preferablyabout 0.1 to about 10 mg. For other animal species, the correspondingdose as converted per 60 kg body weight can be administered.

(3) Quantification of the Peptide of the Present Invention

The antibody of the present invention is capable of specificallyrecognizing the peptide of the present invention, and can thus be usedfor quantification of the peptide of the present invention in a samplefluid, in particular, for quantification by sandwich immunoassay.

That is, the present invention provides:

(i) a method for quantification of the peptide of the present inventionin a sample fluid, which comprises competitively reacting the antibodyof the present invention with a sample fluid and a labeled form of thepeptide of the present invention, and measuring a ratio of the labeledpeptide of the present invention bound to the antibody; and,

(ii) a method for quantification of the peptide of the present inventionin a sample fluid, which comprises simultaneously or continuouslyreacting the sample fluid with the antibody of the present invention anda labeled form of another antibody of the present invention immobilizedon an insoluble carrier, and measuring the activity of the labelingagent on the immobilized carrier.

In the method of quantification (ii) described above, it is preferredthat one antibody is capable of recognizing the N-terminal region of thepeptide of the present invention, while another antibody is capable ofrecognizing the C-terminal region of the peptide of the presentinvention.

The monoclonal antibody to the peptide of the present invention may beused to quantify the peptide of the present invention. Moreover, thepeptide of the present invention may also be detected by means of atissue staining, etc. For these purposes, the antibody molecule per semay be used, or F(ab′)₂, Fab′ or Fab fractions of the antibody moleculemay be used as well.

The method of quantifying the peptide of the present invention using theantibody of the present invention is not particularly limited, and anymethod may be used so far as it relates to a method, in which the amountof an antibody, antigen or antibody-antigen complex can be detected by achemical or a physical means, depending on or corresponding to theamount of antigen (e.g., the amount of the peptide) in a sample fluid tobe assayed, and then calculated using a standard curve prepared by astandard solution containing the known amount of antigen. Advantageouslyused are, for example, nephrometry, competitive method, immunometricmethod and sandwich method; in terms of sensitivity and specificity, thesandwich method, which will be described later, is particularlypreferred.

Examples of labeling agents, which are employed for the assay methodusing the same, are radioisotopes, enzymes, fluorescent substances,luminescent substances, etc. Examples of radioisotopes are [¹²⁵I],[¹³¹I], [³H], [¹⁴C], etc. Preferred examples of enzymes are those thatare stable and have a high specific activity, which includeβ-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, etc. Examples of fluorescent substances arefluorescamine, fluorescein isothiocyanate, etc. Examples of luminescentsubstances are luminol, a luminol derivative, luciferin, lucigenin, etc.Furthermore, a biotin-avidin system may be used as well for binding anantibody or antigen to a labeling agent.

In the immobilization of antigens or antibodies, physical adsorption maybe used. Alternatively, chemical binding that is conventionally used forimmobilization of proteins, enzymes, etc. may be used as well. Examplesof the carrier include insoluble polysaccharides such as agarose,dextran, cellulose, etc.; synthetic resins such as polystyrene,polyacrylamide, silicone, etc.; or glass; and the like.

In the sandwich method, a sample fluid is reacted with an immobilizedform of the monoclonal antibody of the present invention (primaryreaction), then reacted with a labeled form of the monoclonal antibodyof the present invention (secondary reaction) and the activity of thelabeling agent on the insoluble carrier is assayed; thus, the amount ofthe peptide of the present invention in a sample fluid can bedetermined. The primary and secondary reactions may be carried out in areversed order, simultaneously or sequentially with intervals. The typeof the labeling agent and the method of immobilization may be the sameas those described hereinabove. In the immunoassay by the sandwichmethod, it is not always necessary that the antibody used for thelabeled antibody and for the solid phase should be one type or onespecies but a mixture of two or more antibodies may also be used for thepurpose of improving the assay sensitivity, etc.

In the method of assaying the peptide of the present invention by thesandwich method according to the present invention, preferred monoclonalantibodies of the present invention used for the primary and thesecondary reactions are antibodies, which binding sites to the peptideof the present invention are different from each other. Thus, theantibodies used in the primary and secondary reactions are thosewherein, when the antibody used in the secondary reaction recognizes theC-terminal region of the peptide of the present invention, the antibodyrecognizing the site other than the C-terminal regions, e.g.,recognizing the N-terminal region, is preferably used in the primaryreaction.

The monoclonal antibody of the present invention may be used in an assaysystem other than the sandwich method, such as the competitive method,the immunometric method or the nephrometry.

In the competitive method, an antigen in a sample fluid and a labeledantigen are competitively reacted with an antibody, then an unreactedlabeled antigen (F) and a labeled antigen bound to the antibody (B) areseparated (i.e., B/F separation) and the labeled amount of either B or Fis measured to determine the amount of the antigen in the sample fluid.In the reactions for such a method, there are a liquid phase method inwhich a soluble antibody is used as the antibody and the B/F separationis effected by polyethylene glycol, while a second antibody to theantibody is used, and a solid phase method in which an immobilizedantibody is used as the first antibody or a soluble antibody is used asthe first antibody, while an immobilized antibody is used as the secondantibody.

In the immunometric method, an antigen in a sample fluid and animmobilized antigen are competitively reacted with a given amount of alabeled antibody followed by separating the solid phase from the liquidphase; or an antigen in a sample fluid and an excess amount of labeledantibody are reacted, then an immobilized antigen is added to bind anunreacted labeled antibody to the solid phase and the solid phase isseparated from the liquid phase. Thereafter, the labeled amount of anyof the phases is measured to determine the antigen amount in the samplefluid.

In the nephrometry, the amount of insoluble sediment, which is producedas a result of the antigen-antibody reaction in a gel or in a solution,is measured. Even when the amount of an antigen in a sample fluid issmall and only a small amount of the sediment is obtained, a lasernephrometry utilizing laser scattering can be suitably used.

In applying each of those immunoassays to the assay method of thepresent invention, any special conditions, operations, etc. are notrequired. The assay system for the peptide of the present invention maybe constructed in addition to conditions or operations conventionallyused for each of the methods, taking technical consideration by oneskilled in the art into account. For the details of such conventionaltechnical means, a variety of reviews, reference books, etc. may bereferred to:

for example, Hiroshi Irie (ed.): “Radioimmunoassay” (published byKodansha, 1974); Hiroshi Irie (ed.): “Radioimmunoassay; Second Series”(published by Kodansha, 1979); Eiji Ishikawa, et al. (ed.): “EnzymeImmunoassay” (published by Igaku Shoin, 1978); Eiji Ishikawa, et al.(ed.): “Enzyme Immunoassay” (Second Edition) (published by Igaku Shoin,1982); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (Third Edition)(published by Igaku Shoin, 1987); “Methods in Enzymology” Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (all published by Academic Press); etc.

As described above, the peptide of the present invention can bequantified with high sensitivity, using the antibody of the presentinvention.

Furthermore, when a reduced level of the peptide of the presentinvention is detected by quantifying a level of the peptide of thepresent invention using the antibody of the present invention, it can bediagnosed that one suffers from, e.g., anorexia, hypertension,autoimmune disease, heart failure, cataract, glaucoma, acute bacterialmeningitis, acute myocardial infarction, acute pancreatitis, acute viralencephalitis, adult respiratory distress syndrome, alcoholic hepatitis,Alzheimer's disease, asthma, arteriosclerosis, atopic dermatitis,bacterial pneumonia, bladder cancer, fracture, breast cancer, bulimia,polyphagia, burn healing, uterine cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic pancreatitis, livercirrhosis, cancer of the colon and rectum (colon cancer/rectal cancer),Crohn's disease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially, anorexia or the like) etc.; or itis highly likely for one to suffer from these disease in the future.

When an increased level of the peptide of the present invention isdetected, it can be diagnosed that one suffers from, e.g., obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma, Sheehan's syndrome,spermatogenesis disorder (especially, obesity, or the like), etc.; or itis highly likely for one to suffer from these disease in the future.

The antibody of the present invention may also be employed to detect thepeptide of the present invention present in a sample fluid such as bodyfluids, tissues, etc. The antibody may further be used for thepreparation of an antibody column to purify the peptide of the presentinvention, detect the peptide of the present invention in each fractionupon purification, analysis of the behavior of the peptide of thepresent invention in the cells under investigation.

(4) Gene Diagnostic Agent

By using the DNA of the present invention, e.g., as a probe, abnormality(gene abnormality) of the DNA or mRNA encoding the peptide of thepresent invention in human or other warm-blooded animal (e.g., rat,mouse, guinea pig, rabbit, chicken, sheep, swine, bovine, horse, cat,dog, monkey, etc.), can be detected. Thus, the DNA of the presentinvention is useful as a gene diagnostic agent for the damage to the DNAor mRNA, mutation, a decreased expression or an increased expression, oroverexpression of the DNA or mRNA.

The gene diagnosis described above using the DNA of the presentinvention can be performed by, for example, the publicly known northernhybridization assay or the PCR-SSCP assay (Genomics, 5, 874-879 (1989);Proceedings of the National Academy of Sciences of the United States ofAmerica, 86, 2766-2770 (1989)).

When a decreased expression of mRNA is detected, e.g., by northernhybridization, it can be diagnosed that one is likely to suffer from,for example, anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia or the like) etc.; or itis highly likely for one to suffer from diseases in the future.

When overexpression of mRNA is detected by northern hybridization, itcan be diagnosed that one is likely to suffer from, for example, obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like); or itis highly likely for one to suffer from diseases in the future.

(5) Pharmaceutical Composition Comprising Antisense DNA

The antisense DNA that binds complementarily to the DNA of the presentinvention to inhibit expression of the DNA can be used aspreventive/therapeutic agents for diseases, for example, obesity (e.g.,malignant mastocytosis, exogenous obesity, hyperinsulinar obesity,hyperplasmic obesity, hypophyseal adiposity, hypoplasmic obesity,hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like), etc.

For example, when the antisense DNA is used, the antisense DNA may beadministered directly, or the DNA is inserted into an appropriate vectorsuch as retrovirus vector, adenovirus vector, adenovirus-associatedvirus vector, etc. and then administered in a conventional manner. Theantisense DNA may also be administered as an intact DNA, or preparedinto pharmaceutical preparations together with a physiologicallyacceptable carrier such as an adjuvant to assist its uptake andadministered by gene gun or through a catheter such as a catheter with ahydrogel.

In addition, the antisense DNA may also be employed as anoligonucleotide probe for diagnosis to examine the presence of the DNAof the present invention in tissues or cells and states of itsexpression.

(6) Pharmaceutical Composition Comprising the Antibody of the PresentInvention

The antibody of the present invention having the effect to neutralizethe activity of the peptide of the present invention can be used asdrugs for the prevention/treatment of diseases, for example, obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity, or the like), etc.

The therapeutic/preventive agents for diseases described abovecomprising the antibody of the present invention can be administered tohuman or mammals (e.g., rats, rabbits, sheep, swine, bovine, cats, dogs,monkeys, etc.) orally or parenterally directly as a liquid preparation,or as a pharmaceutical composition in an appropriate preparation form.The dose varies depending on subject to be administered, target disease,conditions, route for administration, etc.; when it is used for thetreatment/prevention of the adult patient with, e.g., obesity, the agentis advantageously administered to the patient through intravenousinjection, normally in a single dose of approximately 0.01 to 20 mg/kgbody weight, preferably about 0.1 to about 10 mg/kg body weight, andmore preferably about 0.1 to about 5 mg/kg body weight, approximately 1to 5 times, preferably approximately 1 to 3 times, per day. For otherparenteral administration and oral administration, the correspondingdose may be administered. When the conditions are extremely serious, thedose may be increased depending on the conditions.

The antibody of the present invention may be administered directly as itis or as an appropriate pharmaceutical composition. The pharmaceuticalcomposition used for the administration described above contains apharmacologically acceptable carrier with the aforesaid compounds orsalts thereof, a diluent or excipient. Such a composition is provided inthe preparation suitable for oral or parenteral administration.

That is, examples of the composition for oral administration includesolid or liquid preparations, specifically, tablets (including drageesand film-coated tablets), pills, granules, powdery preparations,capsules (including soft capsules), syrup, emulsions, suspensions, etc.Such a composition is manufactured by publicly known methods andcontains a vehicle, a diluent or excipient conventionally used in thefield of pharmaceutical preparations. Examples of the vehicle orexcipient for tablets are lactose, starch, sucrose, magnesium stearate,etc.

Examples of the composition for parenteral administration that can beused are injections, suppositories, etc. and the injections include theform of intravenous, subcutaneous, transcutaneous, intramuscular anddrip injections, etc. Such injections are prepared by publicly knownmethods, e.g., by dissolving, suspending or emulsifying the aforesaidantibody or its salts in a sterile aqueous or oily liquid medium. Forthe aqueous medium for injection, for example, physiological saline andisotonic solutions containing glucose and other adjuvant, etc. are used.Appropriate dissolution aids, for example, alcohol (e.g., ethanol),polyalcohol (e.g., propylene glycol or polyethylene glycol), nonionicsurfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)] may be used in combination. For theoily solution, for example, sesame oil, soybean oil and the like areused, and dissolution aids such as benzyl benzoate, benzyl alcohol, etc.may be used in combination. The thus-prepared liquid for injection isnormally filled in an appropriate ampoule. The suppository used forrectal administration is prepared by mixing the aforesaid antibody orits salts with conventional suppository base.

The oral or parenteral pharmaceutical composition described above isadvantageously prepared in a unit dosage form suitable for the dose ofthe active ingredient. Examples of such unit dosage form includetablets, pills, capsules, injections (ampoules), suppositories, etc. Itis preferred that the antibody described above is contained generally ina dose of 5 to 500 mg per unit dosage form, 5 to 100 mg especially forinjections and 10 to 250 mg for other preparations.

Each composition described above may further contain other activecomponents unless formulation with the antibody causes any adverseinteraction.

(7) DNA Transgenic Animal

The present invention provides a non-human mammal bearing an exogenousDNA encoding the peptide of the present invention (hereinafter merelyreferred to as the exogenous DNA of the present invention) or itsvariant DNA (sometimes simply referred to as the exogenous variant DNAof the present invention).

Thus, the present invention provides:

(i) a non-human mammal bearing the exogenous DNA or its variant DNA;

(ii) the mammal according to (i), wherein the non-human mammal is arodent;

(iii) the mammal according to (ii), wherein the rodent is mouse or rat;and,

(iv) a recombinant vector bearing the exogenous DNA of the presentinvention or its variant DNA and capable of expressing in a mammal.

The non-human mammal bearing the exogenous DNA of the present inventionor its variant DNA (hereinafter simply referred to as the DNA transgenicanimal of the present invention) can be prepared by transfecting adesired DNA into an unfertilized egg, a fertilized egg, a spermatozoon,a germinal cell containing a primordial germinal cell thereof, or thelike, preferably in the embryogenic stage in the development of anon-human mammal (more preferably in the single cell or fertilized cellstage and generally before the 8-cell phase), by standard means, such asthe calcium phosphate method, the electric pulse method, the lipofectionmethod, the agglutination method, the microinjection method, theparticle gun method, the DEAE-dextran method etc. Also, it is possibleto transfect the exogenous DNA of the present invention into a somaticcell, a living organ, a tissue cell, or the like by the DNA transfectionmethods, and utilize the transformant for cell culture, tissue culture,etc. In addition, these cells may be fused with the above-describedgerminal cell by a publicly known cell fusion method to create thetransgenic animal of the present invention.

Examples of the non-human mammal that can be used include bovine, swine,sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats,and the like. Above all, preferred are rodents, especially mice (e.g.,C57BL/6 strain, DBA2 strain, etc. for a pure line and for a cross line,B6C3F₁ strain, BDF₁ strain B6D2F₁ strain, BALB/c strain, ICR strain,etc.) or rats (Wistar, SD, etc.), since they are relatively short inontogeny and life cycle from a standpoint of creating model animals forhuman disease.

“Mammals” in a recombinant vector that can be expressed in the mammalsinclude the aforesaid non-human mammals and human.

The exogenous DNA of the present invention refers to the DNA of thepresent invention that is once isolated/extracted from mammals, not theDNA of the present invention inherently possessed by the non-humanmammals.

The variant DNA of the present invention includes variants resultingfrom variation (e.g., mutation, etc.) in the base sequence of theoriginal DNA of the present invention, specifically DNAs resulting frombase addition, deletion, substitution with other bases, etc. and furtherincluding abnormal DNA.

The abnormal DNA is intended to mean such a DNA that expresses theabnormal peptide of the present invention and exemplified by the DNAthat expresses a peptide to suppress the functions of the normal peptideof the present invention, or the like.

The exogenous DNA of the present invention may be any one of thosederived from a mammal of the same species as, or a different speciesfrom, the mammal as the target animal. In transfecting the DNA of thepresent invention, it is generally advantageous to use the DNA as a DNAconstruct in which the DNA is ligated downstream a promoter capable ofexpressing the DNA in the target animal. For example, in the case oftransfecting the human DNA of the present invention, a DNA transgenicmammal that expresses the DNA of the present invention to a high level,can be prepared by microinjecting a DNA construct (e.g., vector, etc.)ligated with the human DNA of the present invention into a fertilizedegg of the target non-human mammal downstream various promoters, whichare capable of expressing the DNA derived from various mammals (e.g.,rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) bearingthe DNA of the present invention highly homologous to the human DNA.

As expression vectors for the peptide of the present invention, thereare Escherichia coli-derived plasmids, Bacillus subtilis-derivedplasmids, yeast-derived plasmids, bacteriophages such as λ phage,retroviruses such as Moloney leukemia virus, etc., and animal virusessuch as vaccinia virus, baculovirus, etc. Of these vectors, Escherichiacoli-derived plasmids, Bacillus subtilis-derived plasmids, oryeast-derived plasmids, etc. are preferably used.

Examples of these promoters for regulating the DNA expression describedabove include (1) promoters for the DNA derived from viruses (e.g.,simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breastcancer virus, poliovirus, etc.), and (2) promoters derived from variousmammals (human, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice,etc.), for example, promoters of albumin, insulin II, uroplakin II,elastase, erythropoietin, endothelin, muscular creatine kinase, glialfibrillary acidic protein, glutathione S-transferase, platelet-derivedgrowth factor, keratins K1, K10 and K14, collagen types I and II, cyclicAMP-dependent protein kinase βI subunit, dystrophin, tartarate-resistantalkaline phosphatase, atrial natriuretic factor, endothelial receptortyrosine kinase (generally abbreviated as Tie2), sodium-potassiumadenosine triphosphorylase (Na,K-ATPase), neurofilament light chain,metallothioneins I and IIA, metalloproteinase I tissue inhibitor, MHCclass I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroidperoxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, αand β myosin heavy chains, myosin light chains 1 and 2, myelin baseprotein, thyroglobulins, Thy-1, immunoglobulins, H-chain variable region(VNP), serum amyloid component P, myoglobin, troponin C, smooth muscle aactin, preproencephalin A, vasopressin, etc. Among others them,cytomegalovirus promoters, human peptide elongation factor 1α (EF-1α)promoters, human and chicken β actin promoters etc., which protein canhighly express in the whole body are preferred.

It is preferred that the vectors described above have a sequence forterminating the transcription of the desired messenger RNA in the DNAtransgenic animal (generally called a terminator); for example, asequence of each DNA derived from viruses and various mammals. SV40terminator of simian virus, etc. are preferably used.

In addition, for the purpose of increasing the expression of the desiredexogenous DNA to a higher level, the splicing signal and enhancer regionof each DNA, a portion of the intron of an eukaryotic DNA may also beligated at the 5′ upstream of the promoter region, or between thepromoter region and the translational region, or at the 3′ downstream ofthe translational region, depending upon purpose.

The normal translational region for the peptide of the present inventioncan be obtained using as a starting material the entire genomic DNA orits portion of liver, kidney, thyroid cell or fibroblast origin fromhuman or various mammals (e.g., rabbits, dogs, cats, guinea pigs,hamsters, rats, mice, etc.) or of various commercially available genomicDNA libraries, or using complementary DNA prepared by a publicly knownmethod from RNA of liver, kidney, thyroid cell or fibroblast origin as astarting material. Also, an exogenous abnormal DNA can produce atranslational region, which is obtained by point mutagenesis variationof the normal translational region for a peptide obtained from the cellsor tissues described above.

The said translational region can be prepared by a conventional DNAengineering technique, in which the DNA is ligated downstream theaforesaid promoter and if desired, upstream the translation terminationsite, as a DNA construct capable of being expressed in the transgenicanimal.

The exogenous DNA of the present invention is transfected at thefertilized egg cell stage in a manner such that the DNA is certainlypresent in all the germinal cells and somatic cells of the targetmammal. The fact that the exogenous DNA of the present invention ispresent in the germinal cells of the animal prepared by DNA transfectionmeans that all offspring of the prepared animal will maintain theexogenous DNA of the present invention in all of the germinal cells andsomatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention also have the exogenous DNA inall of the germinal cells and somatic cells thereof.

The non-human mammal, in which the normal exogenous DNA of the presentinvention has been transfected, can be passaged as the DNA-bearinganimal under ordinary rearing environment, by confirming that theexogenous DNA is stably retained by mating.

By the transfection of the exogenous DNA of the present invention at thefertilized egg cell stage, the DNA is retained to be excess in all ofthe germinal and somatic cells. The fact that the exogenous DNA of thepresent invention is excessively present in the germinal cells of theprepared animal after transfection means that the exogenous DNA of thepresent invention is excessively present in all of the germinal cellsand somatic cells thereof. The offspring of the animal that inherits theexogenous DNA of the present invention have excessively the exogenousDNA of the present invention in all of the germinal cells and somaticcells thereof.

By obtaining a homozygotic animal having the transfected DNA in both ofhomologous chromosomes and mating a male and female of the animal, alloffspring can be passaged to retain the DNA.

In a non-human mammal bearing the normal DNA of the present invention,the normal DNA of the present invention has expressed to a high level,and may eventually develop the hyperfunction of the peptide of thepresent invention by promoting the function of endogenous normal DNA.Therefore, the animal can be utilized as a pathologic model animal forsuch a disease. Specifically, using the normal DNA transgenic animal ofthe present invention, it is possible to elucidate the mechanism of thehyperfunction of the peptide of the present invention and thepathological mechanism of the disease associated with the peptide of thepresent invention and to determine how to treat the disease.

Furthermore, since a mammal wherein the exogenous normal DNA of thepresent invention is transfected exhibits an increasing symptom of thepeptide of the present invention librated, the animal is usable forscreening therapeutic agents for the disease associated with the peptideof the present invention.

On the other hand, a non-human mammal having the exogenous abnormal DNAof the present invention can be passaged under normal breedingconditions as the DNA-bearing animal by confirming the stable retainingof the exogenous DNA via crossing. Further, the exogenous DNA to besubjected can be utilized as a starting material by inserting the DNAinto the plasmid described above. The DNA construct with promoter can beprepared by conventional DNA engineering techniques. The transfection ofthe abnormal DNA of the present invention at the fertilized egg cellstage is preserved to be present in all of the germinal and somaticcells of the mammals to be subjected. The fact that the abnormal DNA ofthe present invention is present in the germinal cells of the animalafter DNA transfection means that all of the offspring of the preparedanimal have the abnormal DNA of the present invention in all of thegerminal and somatic cells. Such an offspring passaged the exogenous DNAof the present invention contains the abnormal DNA of the presentinvention in all of the germinal and somatic cells. A homozygous animalhaving the introduced DNA on both of homologous chromosomes can beacquired and then by mating these male and female animals, all theoffspring can be bled to have the DNA.

Since a non-human mammal having the abnormal DNA of the presentinvention may express the abnormal DNA of the present invention at ahigh level, the animal may be the function inactivation typeinadaptability of the peptide of the present invention by inhibiting thefunction of the endogenous normal DNA and can be utilized as its diseasemodel animal. For example, using the abnormal DNA-transgenic animal ofthe present invention, it is possible to elucidate the mechanism ofinadaptability of the peptide of the present invention and to perform tostudy a method for treatment of this disease.

More specifically, the transgenic animal of the present inventionexpressing the abnormal DNA of the present invention to a high level isalso expected to serve as an experimental model for the elucidation ofthe mechanism of the functional inhibition (dominant negative effect) ofnormal peptide by the abnormal peptide of the present invention in thefunction inactive type inadaptability of the peptide of the presentinvention.

A mammal bearing the abnormal exogenous DNA of the present invention isalso expected to serve for screening a candidate drug for the treatmentof the function inactive type inadaptability of the peptide of thepresent invention, since the peptide of the present invention isincreased in such an animal in its free form.

Other potential applications of two kinds of the transgenic animalsdescribed above include:

(1) use as a cell source for tissue culture;

(2) elucidation of the relation to a peptide that is specificallyexpressed or activated by the peptide of the present invention, bydirect analysis of DNA or RNA in tissue of the DNA transgenic animal ofthe present invention or by analysis of the peptide tissue expressed bythe DNA;

(3) research in the function of cells derived from tissues that arecultured usually only with difficulty, using cells of tissue bearing theDNA cultured by a standard tissue culture technique;

(4) screening of a drug that enhances the functions of cells using thecells described in (3) above; and,

(5) isolation and purification of the variant peptide of the presentinvention and preparation of an antibody thereto.

Furthermore, clinical conditions of a disease associated with thepeptide of the present invention, including the function inactive typeinadaptability of the peptide of the present invention can be determinedusing the DNA transgenic animal of the present invention. Also,pathological findings on each organ in a disease model associated withthe peptide of the present invention can be obtained in more detail,leading to the development of a new method for treatment as well as theresearch and therapy of any secondary diseases associated with thedisease.

It is also possible to obtain a free DNA-transfected cell by withdrawingeach organ from the DNA transgenic animal of the present invention,mincing the organ and degrading with a proteinase such as trypsin, etc.,followed by establishing the line of culturing or cultured cells.Furthermore, the DNA transgenic animal of the present invention canserve as identification of cells capable of producing the peptide of thepresent invention, and as studies on association with apoptosis,differentiation or propagation or on the mechanism of signaltransduction in these properties to inspect any abnormality therein.Thus, the DNA transgenic animal of the present invention can provide aneffective research material for the peptide of the present invention andfor elucidating the function and effect thereof.

To develop a therapeutic drug for the treatment of diseases associatedwith the peptide of the present invention, including the functioninactive type inadaptability of the peptide of the present invention,using the DNA transgenic animal of the present invention, an effectiveand rapid method for screening can be provided by using the method forinspection and the method for quantification, etc. described above. Itis also possible to investigate and develop a method for DNA therapy forthe treatment of diseases associated with the peptide of the presentinvention, using the DNA transgenic animal of the present invention or avector capable of expressing the exogenous DNA of the present invention.

(8) Knockout Animal

The present invention provides a non-human mammal embryonic stem cellbearing the DNA of the present invention inactivated and a non-humanmammal deficient in expressing the DNA of the present invention.

Thus, the present invention provides:

(i) a non-human embryonic stem cell in which the DNA of the presentinvention is inactivated;

(ii) an embryonic stem cell according to (i), wherein the DNA isinactivated by introducing a reporter gene (e.g., β-galactosidase genederived from Escherichia coli);

(iii) an embryonic stem cell according to (i), which is resistant toneomycin;

(iv) an embryonic stem cell according to (i), wherein the non-humanmammal is a rodent;

(v) an embryonic stem cell according to (iv), wherein the rodent ismouse;

(vi) a non-human mammal deficient in expressing the DNA of the presentinvention, wherein the DNA of the present invention is inactivated;

(vii) a non-human mammal according to (vi), wherein the DNA isinactivated by inserting a reporter gene (e.g., β-galactosidase derivedfrom Escherichia coli) therein and the reporter gene is capable of beingexpressed under control of a promoter for the DNA of the presentinvention;

(viii) a non-human mammal according to (vi), which is a rodent;

(ix) a non-human mammal according to (viii), wherein the rodent ismouse; and,

(x) a method of screening a compound or its salt that promotes orinhibits the promoter activity for the DNA of the present invention,which comprises administering a test compound to the mammal of (vii) anddetecting expression of the reporter gene.

The non-human mammal embryonic stem cell in which the DNA of the presentinvention is inactivated refers to a non-human mammal embryonic stemcell that suppresses the ability of the non-human mammal to express theDNA by artificially mutating the DNA of the present invention, or theDNA has no substantial ability to express the peptide of the presentinvention (hereinafter sometimes referred to as the knockout DNA of thepresent invention) by substantially inactivating the activity of thepeptide of the present invention encoded by the DNA (hereinafter merelyreferred to as ES cell).

As the non-human mammal, the same examples as described above apply.

Techniques for artificially mutating the DNA of the present inventioninclude deletion of a part or all of the DNA sequence and insertion ofor substitution with other DNA, by genetic engineering. By thesevariations, the knockout DNA of the present invention may be prepared,for example, by shifting the reading frame of a codon or by disruptingthe function of a promoter or exon.

Specifically, the non-human mammal embryonic stem cell in which the DNAof the present invention is inactivated (hereinafter merely referred toas the ES cell with the DNA of the present invention inactivated or theknockout ES cell of the present invention) can be obtained by, forexample, isolating the DNA of the present invention that the desirednon-human mammal possesses, inserting a DNA fragment having a DNAsequence constructed by inserting a drug resistant gene such as aneomycin resistant gene or a hygromycin resistant gene, or a reportergene such as lacZ (β-galactosidase gene) or cat (chloramphenicolacetyltransferase gene), etc. into its exon site thereby to disable thefunctions of exon, or integrating to a chromosome of the subject animalby, e.g., homologous recombination, a DNA sequence which terminates genetranscription (e.g., polyA additional signal, etc.) in the intronbetween exons, thus inhibiting the synthesis of complete messenger RNAto eventually destroy the gene (hereinafter simply referred to astargeting vector). The thus obtained ES cells are subjected to Southernhybridization analysis using a DNA sequence on or near the DNA of thepresent invention as a probe, or to PCR analysis using a DNA sequence onthe targeting vector and another DNA sequence near the DNA of thepresent invention, which is not included in the targeting vector asprimers, thereby to select the knockout ES cell of the presentinvention.

The parent ES cells to inactivate the DNA of the present invention byhomologous recombination, etc. may be of a strain already established asdescribed above, or may be originally established in accordance with amodification of the known method by Evans and Kaufman supra. Forexample, in the case of mouse ES cells, currently it is common practiceto use ES cells of the 129 strain. However, since their immunologicalbackground is obscure, the C57BL/6 mouse or the BDF₁ mouse (F₁ hybridbetween C57BL/16 and DBA/2), wherein the low ovum availability perC57BL/6 in the C57B116 mouse has been improved by crossing with DBA/2,may be preferably used, instead of obtaining a pure line of ES cellswith the clear immunological genetic background and for other purposes.The BDF₁ mouse is advantageous in that, when a pathologic model mouse isgenerated using ES cells obtained therefrom, the genetic background canbe changed to that of the C57BL/6 mouse by back-crossing with theC57BL/6 mouse, since its background is of the C57BL/6 mouse, as well asbeing advantageous in that ovum availability per animal is high and ovaare robust.

In establishing ES cells, blastocytes at 3.5 days after fertilizationare commonly used. In the present invention, embryos are preferablycollected at the 8-cell stage, after culturing until the blastocytestage, the embryos are used to efficiently obtain a large number ofearly stage embryos.

Although the ES cells used may be of either sex, male ES cells aregenerally more convenient for generation of a germ cell line chimera andare therefore preferred. It is also desirable that sexes are identifiedas soon as possible to save painstaking culture time.

Methods for sex identification of the ES cell include the method inwhich a gene in the sex-determining region on the Y-chromosome isamplified by the PCR process and detected. When this method is used, onecolony of ES cells (about 50 cells) is sufficient for sex-determinationanalysis, which karyotype analysis, for example G-banding method,requires about 10⁶ cells; therefore, the first selection of ES cells atthe early stage of culture can be based on sex identification, and malecells can be selected early, which saves a significant amount of time atthe early stage of culture.

Second selection can be achieved by, for example, number of chromosomeconfirmation by the G-banding method. It is usually desirable that thechromosome number of the obtained ES cells be 100% of the normal number.However, when it is difficult to obtain the cells having the normalnumber of chromosomes due to physical operation etc. in cellestablishment, it is desirable that the ES cell be again cloned to anormal cell (e.g., in mouse cells having the number of chromosomes being2n=40) after the gene of the ES cells is rendered knockout.

Although the embryonic stem cell line thus obtained shows a very highgrowth potential, it must be subcultured with great care, since it tendsto lose its ontogenic capability. For example, the embryonic stem cellline is cultured at about 37° C. in a carbon dioxide incubator(preferably about 5% carbon dioxide and about 95% air, or about 5%oxygen, about 5% carbon dioxide and 90% air) in the presence of LIF(1-10000 U/ml) on appropriate feeder cells such as STO fibroblasts,treated with a trypsin/EDTA solution (normally about 0.001 to about 0.5%trypsin/about 0.1 to about 5 mM EDTA, preferably about 0.1% trypsin/1 mMEDTA) at the time of passage to obtain separate single cells, which arethen seeded on freshly prepared feeder cells. This passage is normallyconducted every 1 to 3 days; it is desirable that cells are observed atpassage and cells found to be morphologically abnormal in culture, ifany, should be abandoned.

Where ES cells are allowed to reach a high density in mono-layers or toform cell aggregates in suspension under appropriate conditions, theywill spontaneously differentiate to various cell types, for example,pariental and visceral muscles, cardiac muscle or the like (M. J. Evansand M. H. Kaufman, Nature, 292, 154, 1981; G. R. Martin, Proc. Natl.Acad. Sci. U.S.A., 78, 7634, 1981; T. C. Doetschman et al., Journal ofEmbryology Experimental Morphology, 87, 27, 1985). The cells deficientin expressing the DNA of the present invention, which are obtainablefrom the differentiated ES cells of the present invention, are usefulfor studying the peptide of the present invention or the receptorprotein of the present invention from an aspect of cell biology.

The non-human mammal deficient in expressing the DNA of the presentinvention can be identified from a normal animal by measuring the mRNAamount in the subject animal by a publicly known method, and indirectlycomparing the levels of expression.

As the non-human mammal, the same examples as described above apply.

With respect to the non-human mammal deficient in expressing the DNA ofthe present invention, the DNA of the present invention can be renderedknockout by transfecting a targeting vector, prepared as describedabove, to non-human mammal embryonic stem cells or oocytes thereof, andconducting homologous recombination in which a targeting vector DNAsequence, wherein the DNA of the present invention is inactivated by thetransfection, is replaced with the DNA of the present invention on achromosome of a non-human mammal embryonic stem cell or embryo thereof.

The cells with the DNA of the present invention knockout can beidentified by Southern hybridization analysis using a DNA sequence on ornear the DNA of the present invention as a probe, or by PCR analysisusing as primers a DNA sequence on the targeting vector and another DNAsequence, which is not included in the targeting vector. When non-humanmammalian embryonic stem cells are used, a cell line wherein the DNA, ofthe present invention is inactivated by homologous recombination iscloned; the resulting cloned cell line is injected to, e.g., a non-humanmammalian embryo or blastocyte, at an appropriate stage such as the8-cell stage. The resulting chimeric embryos are transplanted to theuterus of the pseudopregnant non-human mammal. The resulting animal is achimeric animal composed of both cells having the normal locus of theDNA of the present invention and those having an artificially mutatedlocus of the DNA of the present invention.

When some germ cells of the chimeric animal have a mutated locus of theDNA of the present invention, an individual, which entire tissue iscomposed of cells having a mutated locus of the DNA of the presentinvention can be selected from a series of offspring obtained bycrossing between such a chimeric animal and a normal animal, e.g., bycoat color identification, etc. The individuals thus obtained arenormally deficient in heterozygous expression of the peptide of thepresent invention. The individuals deficient in homozygous expression ofthe peptide of the present invention or the receptor protein of thepresent invention can be obtained from offspring of the intercrossbetween the heterozygotes of the peptide of the present invention or thereceptor protein of the present invention.

When an oocyte or egg cell is used, a DNA solution may be injected,e.g., to the prenucleus by microinjection thereby to obtain a transgenicnon-human mammal having a targeting vector introduced in a chromosomethereof. From such transgenic non-human mammals, those having a mutationat the locus of the DNA of the present invention can be obtained byselection based on homologous recombination.

As described above, individuals in which the DNA of the presentinvention is rendered knockout permit passage rearing under ordinaryrearing conditions, after the individuals obtained by their crossinghave proven to have been knockout.

Furthermore, the genital system may be obtained and maintained byconventional methods. That is, by crossing male and female animals eachhaving the inactivated DNA, homozygote animals having the inactivatedDNA in both loci can be obtained. The homozygotes thus obtained may bereared so that one normal animal and two or more homozygotes areproduced from a mother animal to efficiently obtain such homozygotes. Bycrossing male and female heterozygotes, homozygotes and heterozygoteshaving the inactivated DNA are proliferated and passaged.

The non-human mammal embryonic stem cell, in which the DNA of thepresent invention is inactivated, is very useful for preparing anon-human mammal deficient in expressing the DNA of the presentinvention.

Since the non-human mammal deficient in expressing the DNA of thepresent invention lacks various biological activities derived from thepeptide of the present invention, such an animal can be a disease modelsuspected of inactivated biological activities of the peptide of thepresent invention and thus, offers an effective study to investigatecauses for and therapy for these diseases.

(8a) Method of Screening Compounds having Therapeutic/Preventive Effectson Diseases caused by Deficiency, Damages, etc. of the DNA of thePresent Invention

The non-human mammal deficient in expressing the DNA of the presentinvention can be employed for screening of compounds havingtherapeutic/prophylactic effects on diseases caused by deficiency,damages, etc. of the DNA of the present invention.

That is, the present invention provides a method for screening of acompound or its salt having therapeutic/preventive effects on diseasescaused by deficiency, damages, etc. of the DNA of the present invention,which comprises administering a test compound to the non-human mammaldeficient in expressing the DNA of the present invention andobserving/measuring a change occurred in the animal.

As the non-human mammal deficient in expressing the DNA of the presentinvention which can be employed for the screening method, the sameexamples as given hereinabove apply.

Examples of the test compounds include peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts,vegetable extracts, animal tissue extracts, blood plasma, etc. Thesecompounds may be novel compounds or publicly known compounds.

Specifically, the non-human mammal deficient in expressing the DNA ofthe present invention is treated with a test compound, comparison ismade with an intact animal for control and a change in each organ,tissue, disease conditions, etc. of the animal is used as an indicatorto assess the therapeutic/prophylactic effects of the test compound.

For treating an animal to be test with a test compound, for example,oral administration, intravenous injection, etc. are applied and thetreatment is appropriately selected depending upon conditions of thetest animal, properties of the test compound, etc. Furthermore, a doseof test compound to be administered can be appropriately chosendepending on method for administration, nature of the test compound,etc.

In screening compounds having the therapeutic/preventive effect on,e.g., anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress-syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia, or the like), thenon-human mammal deficient in expressing the DNA of the presentinvention is subjected to a sugar loading treatment, a test compound isadministered before or after the sugar loading treatment and, bloodsugar level, body weight change, etc. of the animal is measured withpassage of time.

In the screening method described above, when a test compound isadministered to a test animal and found to reduce the blood sugar levelof the animal to at least about 10%, preferably at least about 30% andmore preferably at least 25 about 50%, the test compound can be selectedto be a compound having a therapeutic/preventive effect on the diseasesabove.

The compound obtained using the screening method above is a compoundselected from the test compounds described, above and exhibits atherapeutic/preventive effect on the diseases caused by deficiencies,damages, etc. of the peptide of the present invention. Therefore, thecompound can be employed as a safe and low toxic drug for the treatmentand prevention of these diseases. Furthermore, compounds derived fromsuch a compound obtained by the screening described above can besimilarly employed.

The compound obtained by the screening method above may be in the formof salts. As such salts, there may be used salts with physiologicallyacceptable acids (e.g., inorganic acids, organic acids, etc.) or bases(e.g., alkali metal salts, etc.), preferably in the form ofphysiologically acceptable acid addition salts. Examples of such saltsare salts with inorganic acids (e.g., hydrochloric acid, phosphoricacid, hydrobromic acid, sulfuric acid, etc.), salts with organic acids(e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.)and the like.

A pharmaceutical composition comprising the compound or its salt,obtained by the above screening method, may be manufactured in a mannersimilar to the method for preparing the pharmaceutical compositioncomprising the peptide of the present invention described hereinabove.

Since the pharmaceutical composition thus obtained is safe and lowtoxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

A dose of the compound or its salt to be administered varies dependingupon particular disease, subject to be administered, route ofadministration, etc., and in oral administration to an adult patientwith anorexia (as 60 kg body weight), the compound is administeredgenerally in a dose of approximately 0.1 to 100 mg, preferablyapproximately 1.0 to 50 mg, more preferably approximately 1.0 to 20 mgper day. For parenteral administration to an adult patient with anorexia(as 60 kg body weight), it is advantageous to administer the compoundintravenously in the form of an injectable preparation in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day, though the single dosagevaries depending upon particular subject, particular disease, etc. Forother animals, the compound can be administered in the correspondingdose with converting it into that for the 60 kg body weight.

(8b) Method of Screening a Compound that Promotes or Inhibits theActivity of a Promoter to the DNA of the Present Invention

The present invention provides a method of screening a compound or itssalt that promotes or inhibits the activity of a promoter to the DNA ofthe present invention, which comprises administering a test compound toa non-human mammal deficient in expressing the DNA of the presentinvention and detecting expression of the reporter gene.

In the screening method described above, the non-human mammal deficientin expressing the DNA of the present invention is selected from theaforesaid non-human mammal deficient in expressing the DNA of thepresent invention, as an animal in which the DNA of the presentinvention is inactivated by introducing a reporter gene and the reportergene is expressed under control of a promoter to the DNA of the presentinvention.

The same examples of the test compound apply to those given above.

As the reporter gene, the same specific examples apply. Preferablyemployed are β-galactosidase (lacZ), soluble alkaline phosphatase gene,luciferase gene and the like.

Since the reporter gene is present under control of a promoter to theDNA of the present invention in the non-human mammal deficient inexpressing the DNA of the present invention wherein the DNA of thepresent invention is substituted with the reporter gene, the activity ofthe promoter can be detected by tracing expression of a substanceencoded by the reporter gene.

For example, when a part of the DNA region encoding the peptide of thepresent invention is substituted with, e.g., β-galactosidase gene (lacZ)derived from Escherichia coli, β-galactosidase is expressed in a tissuewhere the peptide of the present invention should originally beexpressed, instead of the peptide of the present invention. Thus, thestate of expression condition of the peptide of the present inventioncan be readily observed in vivo of an animal by staining with a reagent,e.g., 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) which issubstrate for β-galactosidase. Specifically, a mouse deficient in thepeptide of the present invention, or its tissue slice section is fixedwith glutaraldehyde, etc. After washing with phosphate buffered saline(PBS), the system is reacted with a staining solution containing X-galat room temperature or about 37° C. for approximately 30 minutes to anhour. After the β-galactosidase reaction is terminated by washing thetissue preparation with 1 mM EDTA/PBS solution, the color formed isobserved. Alternatively, mRNA encoding lacZ may be detected in aconventional manner.

The compound or salts thereof obtained using the aforesaid screeningmethod are compounds that are selected from the test compounds describedabove and the compounds that promote or inhibit the activity of apromoter to the DNA of the present invention.

The compound obtained by the screening method above may form salts. Assalts of the compound, there may be used salts with physiologicallyacceptable acids (e.g., inorganic acids, etc.) or bases (e.g., organicacids, etc.), and especially preferred are physiologically acceptableacid addition salts. Examples of such salts are salts with inorganicacids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid,sulfuric acid, etc.), salts with organic acids (e.g., acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid, etc.) and the like.

The compound or its salt that promotes the promoter activity to the DNAof the present invention can promote expression of the peptide of thepresent invention thereby to promote the function of the peptide. Thus,these compounds are useful as safe and low-toxic drugs for thetreatment/prevention of diseases, e.g., anorexia, hypertension,autoimmune disease, heart failure, cataract, glaucoma, acute bacterialmeningitis, acute myocardial infarction, acute pancreatitis, acute viralencephalitis, adult respiratory distress syndrome, alcoholic hepatitis,Alzheimer's disease, asthma, arteriosclerosis, atopic dermatitis,bacterial pneumonia, bladder cancer, fracture, breast cancer, bulimia,polyphagia, burn healing, uterine cervical cancer, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic pancreatitis, livercirrhosis, cancer of the colon and rectum (colon cancer/rectal cancer),Crohn's disease, dementia, diabetic complications, diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, gastritis, Helicobacterpylori bacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia or the like) orthe like (especially, appetite (eating) stimulant).

The compound or its salt that inhibits the activity of a promoter to theDNA of the present invention can inhibit expression of the peptide ofthe present invention thereby to inhibit the function of the peptide.Thus, these compounds are useful as drugs, includingpreventive/therapeutic drugs (prolactin production inhibitors) fordiseases, for example, obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, pituitary tumor,diencephalon tumor, menstrual disorder, autoimmune disease,prolactinoma, sterility, impotence, amenorrhea, lactorrhea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, lymphoma or Sheehan's syndrome, spermatogenesis disorders,etc.; preferably as preventive/therapeutic agents for obesity,hyperphagia, etc.

Furthermore, compounds derived from the compounds obtained by thescreening described above may be likewise used.

The pharmaceuticals comprising the compound or its salt obtained by thescreening method may be manufactured as in the aforesaid pharmaceuticalscomprising the peptide of the present invention or its salt.

Since the pharmaceutical preparation thus obtained is safe and lowtoxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

A dose of the compound or its salt to be administered varies dependingupon target disease, subject to be administered, route ofadministration, etc.; when the compound that promotes the promoteractivity to the DNA of the present invention is orally administered toan adult patient with, e.g., anorexia (as 60 kg body weight), thecompound is administered generally in a dose of approximately 0.1 to 100mg, preferably approximately 1.0 to 50 mg, more preferably approximately1.0 to 20 mg per day. In parenteral administration, a single dose of thecompound varies depending upon subject to be administered, targetdisease, etc. When the compound that promotes the promoter activity tothe DNA of the present invention is administered to an adult patientwith, e.g., anorexia (as 60 kg body weight) in the form of an injectablepreparation, it is advantageous to administer the compound,intravenously in a dose of approximately 0.01 to 30 mg, preferablyapproximately 0.1 to 20 mg, more preferably approximately 0.1 to 10 mgper day. For other animals, the compound can be administered in thecorresponding dose with converting it into that for the 60 kg bodyweight.

On the other hand, when a compound that inhibits the promoter activityto the DNA of the present invention is orally administered, the compoundis orally administered to an adult patient with, e.g., obesity (as 60 kgbody weight) generally in a dose of approximately 0.1 to 100 mg,preferably approximately 1.0 to 50 mg, more preferably approximately 1.0to 20 mg per day. In parenteral administration, a single dose of thecompound varies depending upon subject to be administered, targetdisease, etc. When the compound that inhibits the promoter activity tothe DNA of the present invention is administered to an adult patientwith, e.g., obesity (as 60 kg body weight) in the form of an injectablepreparation, it is advantageous to administer the compound intravenouslyin a dose of approximately 0.01 to 30 mg, preferably approximately 0.1to 20 mg, more preferably approximately 0.1 to 10 mg per day. For otheranimals, the compound can be administered in the corresponding dose withconverting it into that for the 60 kg body weight.

As described above, the non-human mammal deficient in expressing the DNAof the present invention is extremely useful for screening a compound orits salt that promotes or inhibits the activity of a promoter to the DNAof the present invention, and can thus greatly contribute toinvestigations of causes for various diseases caused by failure toexpress the DNA of the present invention or to development ofpreventive/therapeutic agents for these diseases.

Moreover, when a so-called transgenic animal (gene-transfected animal)is prepared by using a DNA containing the promoter region of the peptideof the present invention, ligating genes encoding various proteinsdownstream the same and injecting the genes into animal oocyte, thepeptide can be specifically synthesized by the animal so that it becomespossible to investigate the activity in vivo. Furthermore, when anappropriate reporter gene is ligated to the promoter region describedabove to establish a cell line so as to express the gene, such can beused as a survey system of low molecular weight compounds thatspecifically promotes or suppresses the ability of producing the peptideitself of the present invention in vivo.

The utilities of bovine GPR7 and bovine GPR8 (hereinafter merelyreferred to as the bovine GPR7/8) of the present invention are explainedbelow.

Antibodies to the bovine GPR7/8 of the present invention (hereinaftersometimes simply referred to as the bovine GPR7/8 antibody) may be anyof polyclonal antibodies and monoclonal antibodies, as long as they arecapable of recognizing antibodies to the bovine GPR7/8 of the presentinvention.

The antibody of the present invention may be manufactured by publiclyknown methods for manufacturing antibodies or antisera, using the bovineGPR7/8 of the present invention as an antigen.

The antisense DNA (hereinafter sometimes merely referred to as thebovine GPR7/8 antisense DNA) having a complementary or substantiallycomplementary base sequence to the DNA (hereinafter sometimes merelyreferred to as the bovine GPR7/8 DNA) encoding the bovine GPR7/8 of thepresent invention can be any antisense DNA, so long as it has a basesequence complementary or substantially complementary to that of thebovine GPR7/8 DNA of the present invention and capable of suppressingexpression of the DNA.

The base sequence substantially complementary to the bovine GPR7/8 DNAof the present invention may, for example, be a base sequence having atleast about 70% homology, preferably at least about 80% homology, morepreferably at least about 90% homology and most preferably at leastabout 95% homology, to the full-length base sequence or partial basesequence of the base sequence complementary to the bovine GPR7/8 DNA ofthe present invention (i.e., complementary strand to the DNA of thepresent invention). In the entire base sequence of the complementarystrand to the bovine GPR7/8 DNA of the present invention, an antisenseDNA having at least about 70% homology, preferably at least about 80%homology, more preferably at least about 90% homology and mostpreferably at least about 95% homology, to the complementary strand ofthe base sequence which encodes the N-terminal region of the bovineGPR7/8 of the present invention (e.g., the base sequence around theinitiation codon) is particularly preferred. These antisense DNAs can besynthesized using a publicly known DNA synthesizer, etc.

The utilities of (1) the bovine GPR7/8 of the present invention, (2) thebovine GPR7/8 DNA of the present invention, (3) the bovine GPR7/8antibody of the present invention, and (4) the bovine GPR7/8 antisenseDNA are explained below.

(1) Therapeutic/Preventive Agent for Diseases with which the BovineGPR7/8 of the Present Invention is Associated

As shown in EXAMPLE 25 later described, the bovine GPR7/8 of the presentinvention is a receptor to the peptide of the present invention.

Accordingly, when the bovine GPR7/8 of the present invention or thebovine GPR7/8 DNA of the present invention involves any abnormality ordeficiency, it is highly likely to cause various diseases, includinganorexia, hypertension, autoimmune disease, heart failure, cataract,glaucoma, acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type I), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative diseases (especially anorexia, etc.), or the like.

Therefore, the bovine GPR7/8 of the present invention and the bovineGPR7/8 DNA of the present invention can be used as pharmaceuticals (inparticular, appetite (eating) stimulants, etc.) for thetreatment/prevention of various diseases as described above (especiallyanorexia, etc.).

When a patient has a reduced level of, or deficient in the bovine GPR7/8of the present invention in his or her body, the bovine GPR7/8 of thepresent invention and the bovine GPR7/8 DNA of the present invention canexhibit the role of the bovine GPR7/8 of the present inventionsufficiently or properly for the patient, (a) by administering thebovine GPR7/8 DNA of the present invention to the patient to express thebovine GPR7/8 of the present invention in the body, (b) by inserting thebovine GPR7/8 DNA of the present invention into a cell, expressing thebovine GPR7/8 of the present invention and then transplanting the cellto the patient, or (c) by administering the bovine GPR7/8 of the presentinvention to the patient, or the like.

Where the bovine GPR7/8 of the present invention or the bovine GPR7/8DNA of the present invention is used as drugs for thetreatment/prevention described above, these drugs can be manufacturedand used similarly to the pharmaceuticals comprising the peptide of thepresent invention or the DNA of the present invention described above.

(2) Screening of Drug Candidate Compounds for Diseases

(2-1) Screening Method A

The method of screening a compound or its salt that alters the bindingof the bovine GPR7/8 of the present invention to the peptide of thepresent invention is carried out as described hereinabove.

(2-2) Screening Method B

Next, the method of screening a compound that regulates the expressionlevel of bovine GPR7/8 is explained below.

The screening method B of the present invention is specifically (i) amethod of screening a compound or its salt that increases or decreasesthe expression level of bovine GPR7/8, which comprises assaying theexpression level of bovine GPR7/8 or the level of mRNA encoding thebovine GPR7/8 in the case that a cell or tissue capable of expressingthe bovine GPR7/8 is cultured in the presence or absence of a testcompound, and comparing the expression level in each case.

As the cell or tissue capable of expressing the bovine GPR7/8, there maybe used a cell or tissue derived from human or other warm-bloodedanimals (e.g., guinea pigs, rats, mice, chicken, rabbits, swine, sheep,bovine, monkeys, etc.); any cell (e.g., nerve cells, endocrine cells,neuroendocrine cells, glial cells, β cells of pancreas, bone marrowcells, hepatocytes, splenocytes, mesangial cells, epidermic cells,epithelial cells, endothelial cells, fibroblasts, fibrocytes, myocytes,fat cells, immune cells (e.g., macrophages, T cells, B cells, naturalkiller cells, mast cells, neutrophils, basophils, eosinophils,monocytes, dendritic, cells), megakaryocyte, synovial cells,chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland cellsor interstitial cells, the corresponding precursor cells, stem cells,cancer cells, etc., of these cells), or any tissue where such a cell ispresent, e.g., brain or any region of the brain (e.g., olfactory bulb,amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus,cerebral cortex, medulla oblongata, cerebellum), spinal cord,hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid,gall-bladder, bone marrow, adrenal gland, skin, muscle, lung,gastrointestinal tract (e.g., large intestine and small intestine),blood vessel, heart, thymus, spleen, submandibular gland, peripheralblood, prostate, testis, ovary, placenta, uterus, bone, cartilage,joint, skeletal muscle, etc., wherein established cell line or primaryculture system may also be used. Transformants transformed by arecombinant vector bearing a DNA encoding the bovine GPR7/8 describedabove may also be used.

To cultivate the cells capable of expressing the bovine GPR7/8, themethod given for cultivating transformants above applies.

As the test compound, a DNA library may also be used, in addition to thetest compounds described above.

The expression level of bovine GPR7/8 can be determined by publiclyknown methods such as immunochemical methods, etc., using an antibody,etc. Alternatively, mRNA encoding the bovine GPR7/8 can be determined bypublicly known methods including northern hybridization, RT-PCR orTaqMan PCR.

Comparison of the expression level of mRNA can be made by publicly knownmethods or a modification thereof, for example, according to the methoddescribed in Molecular Cloning, 2nd (J. Sambrook et al., Cold SpringHarbor Lab. Press, 1989), etc.

Specifically, the level of mRNA encoding the bovine GPR7/8 is determinedby contacting RNA extracted from cells according to publicly knownmethods with the DNA encoding the bovine GPR7/8 or a part thereof or thebovine GPR7/8 antisense polynucleotide of the present invention, andassaying the level of mRNA bound to the DNA encoding the bovine GPR7/8or a part thereof or the antisense polynucleotide of the presentinvention. The level of mRNA bound to the DNA encoding the bovine GPR7/8or a part thereof or the bovine GPR7/8 antisense polynucleotide of thepresent invention can be readily assayed by labeling the DNA encodingthe bovine GPR7/8 or a part thereof or the bovine GPR7/8 antisensepolynucleotide of the present invention with, e.g., a radioisotope, adye, etc. Examples of the radioisotope are ³²P, ³H, etc. Examples of thedye used are fluorescent dyes such as fluorescein, FAM (Biosystems,Inc.), JOE (PE Biosystems, Inc.), TAMRA (PE Biosystems, Inc.), ROX (PEBiosystems, Inc.), Cy5 (Amersham), Cy3 (Amersham), etc.

The level of mRNA can also be determined by converting RNA extractedfrom cells into cDNA by a reverse transcriptase, amplifying the cDNA byPCR using the DNA encoding the bovine GPR7/8 or a part thereof or thebovine GPR7/8 antisense polynucleotide of the present invention as aprimer, and assaying the amount of cDNA amplified.

As described above, the test compound that increases the level of mRNAencoding the bovine GPR7/8 can be selected as a compound that increasesthe expression level of bovine GPR7/8. Also, the test compound thatdecreases the level of mRNA encoding the bovine GPR7/8 can be selectedas a compound that decreases the expression level of bovine GPR7/8.

The present invention further provides:

(ii) a method of screening a compound that promotes or inhibits apromoter activity, which comprises assaying the reporter activity in thecase that a transformant transformed by a recombinant DNA ligated with areporter gene downstream the promoter region or enhancer region of agene encoding the bovine GPR7/8 is cultured in the presence or absenceof a test compound, and comparing the activity in each case.

As the reporter gene, there may be employed, e.g., lacZ (β-galactosidasegene), chloramphenicol acetyltransferase (CAT), luciferase, growthfactor, β-glcuronidase, alkaline phosphatase, green fluorescent protein(GFP), β-lactamase, etc.

By determining the level of the reporter gene product (e.g., mRNA,protein) using publicly known methods, the test compound that increasesthe level of the reporter gene product can be selected as the compoundhaving the activity of regulating (especially promoting) the promoter orenhancer activity of bovine GPR7/8 of the present invention, i.e., thecompound having the activity of increasing the expression level ofbovine GPR7/8. To the contrary, the test compound that decreases thelevel of the reporter gene product can be selected as the compoundhaving the activity of regulating (especially inhibiting) the promoteror enhancer activity of bovine GPR7/8, i.e., the compound having theactivity of decreasing the expression level of bovine GPR7/8.

As the test compounds, those described above are employed.

The transformants can be cultivated as given for the transformantsdescribed above.

Construction of vectors for the reporter genes and assay can beperformed according to publicly known techniques (e.g., MolecularBiotechnology, 13, 29-43, 1999).

The compounds having the activity of increasing the expression level ofbovine GPR7/8 are useful-as safe and low-toxic drugs (e.g.,preventive/therapeutic agents for anorexia, appetite (eating)stimulants, preventive/therapeutic agents for pituitary hormonesecretion disorders [e.g., prolactin secretion disorders (e.g.,hypoovarianism, spermatic underdevelopment, menopausal symptoms,hypothyroidism, etc.)].

The compounds having the activity of decreasing the expression level ofbovine GPR7/8 are useful as safe and low-toxic drugs for theprevention/treatment of obesity (e.g., malignant mastocytosis, exogenousobesity, hyperinsulinar obesity, hyperplasmic obesity, hypophysealadiposity, hypoplasmic obesity, hypothyroid obesity, hypothalamicobesity, symptomatic obesity, infantile obesity, upper body obesity,alimentary obesity, hypogonadal obesity, systemic mastocytosis, simpleobesity, central obesity, etc.), hyperphagia, etc.; as safe andlow-toxic drugs for the prevention/treatment (prolactin productionsuppressing agents) for pituitary tumor, diencephalon tumor, menstrualdisorders, autoimmune disease, prolactinoma, sterility, impotence,amenorrhea, lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-delCastillo syndrome, Forbes-Albright syndrome, lymphoma or Sheehan'ssyndrome, spermatogenesis disorder, etc.; preferably, as safe andlow-toxic drugs for the prevention/treatment of obesity, hyperphagia,etc.

The compound or its salt, which can be obtained by using the screeningmethod B or the screening kit of the present invention, is a compoundselected from, e.g., peptides, proteins, non-peptide compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts, plasma, etc., and is the compound thatpromotes or inhibits the function of the peptide of the presentinvention.

For salts of the compound, those as described for the peptide of thepresent invention are employed.

When the compound obtained by the screening method B or screening kit ofthe present invention is used as the therapeutic/preventive agentdescribed above, the compound can be prepared into pharmaceuticalpreparations and provided for use, in a similar manner to the aforesaidpharmaceuticals comprising the compound or its salt that alters theexpression level of the peptide of the present invention describedabove.

(3) Quantification of the Bovine GPR7/8 of the Present Invention

The antibody of the present invention is capable of specificallyrecognizing the bovine GPR7/8 of the present invention, and can thus beused for quantification of the bovine GPR7/8 of the present invention ina sample fluid, in particular, for quantification by sandwichimmunoassay.

That is, the present invention provides:

(i) a method for quantification of the bovine GPR7/8 of the presentinvention in a sample fluid, which comprises competitively reacting thebovine GPR7/8 antibody of the present invention with a sample fluid anda labeled form of the bovine GPR7/8 of the present invention, andmeasuring a ratio of the labeled bovine GPR7/8 of the present inventionbound to the antibody; and,

(ii) a method for quantification of the bovine GPR7/8 of the presentinvention in a sample fluid, which comprises simultaneously orcontinuously reacting the sample fluid with the antibody of the presentinvention and a labeled form of another antibody of the presentinvention immobilized on an insoluble carrier, and measuring theactivity of the labeling agent on the immobilized carrier.

In the method of quantification (ii) described above, it is preferredthat one antibody is capable of recognizing the N-terminal region of thebovine GPR7/8 of the present invention, while another antibody iscapable of recognizing the C-terminal region of the bovine GPR7/8 of thepresent invention.

The monoclonal antibody to the bovine GPR7/8 of the present inventionmay be used to quantify the bovine GPR7/8 of the present invention, andmay further be used to detect the same by means of a tissue staining,etc. For these purposes, the antibody molecule per se may be used, orF(ab′)₂, Fab′ or Fab fractions of the antibody molecule may be used aswell.

The method of quantifying the bovine GPR7/8 of the present inventionusing the bovine GPR7/8 antibody of the present invention is notparticularly limited, and any method may be used so far as it relates toa method, in which the amount of an antibody, antigen orantibody-antigen complex can be detected by a chemical or a physicalmeans, depending on or corresponding to the amount of antigen (e.g., theamount of the peptide) in a sample fluid to be assayed, and thencalculated using a standard curve prepared by a standard solutioncontaining the known amount of antigen. Advantageously used are, forexample, nephrometry, competitive method, immunometric method andsandwich method; in terms of sensitivity and specificity, the sandwichmethod, which will be described later, is particularly preferred.

Examples of labeling agents, which are employed for the assay methodusing the same, are radioisotopes, enzymes, fluorescent substances,luminescent substances, etc. Examples of radioisotopes are [¹²⁵I],[¹³¹I], [³H], [¹⁴C], etc. Preferred examples of enzymes are those thatare stable and have a high specific activity, which includeβ-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, etc. Examples of fluorescent substances arefluorescamine, fluorescein isothiocyanate, etc. Examples of luminescentsubstances are luminol, a luminol derivative, luciferin, lucigenin, etc.Furthermore, a biotin-avidin system may be used as well for binding anantibody or antigen to a labeling agent.

In the immobilization of antigens or antibodies, physical adsorption maybe used. Alternatively, chemical binding that is conventionally used forimmobilization of proteins, enzymes, etc. may be used as well. Examplesof the carrier include insoluble polysaccharides such as ag arose,dextran, cellulose, etc.; synthetic resins such as polystyrene,polyacrylamide, silicone, etc.; or glass; and the like.

In the sandwich method, a sample fluid is reacted with an immobilizedform of the bovine GPR7/8 monoclonal antibody of the present invention(primary reaction), then reacted with a labeled form of the bovineGPR7/8 monoclonal antibody of the present invention (secondary reaction)and the activity of the labeling agent on the insoluble carrier isassayed; thus, the amount of the bovine GPR7/8 of the present inventionin a sample fluid can be determined. The primary and secondary reactionsmay be carried out in a reversed order, simultaneously or sequentiallywith intervals. The type of the labeling agent and the method ofimmobilization may be the same as those described hereinabove. In theimmunoassay by the sandwich method, it is not always necessary that theantibody used for the labeled antibody and for the solid phase should beone type or one species but a mixture of two or more antibodies may alsobe used for the purpose of improving the assay sensitivity, etc.

In the method of assaying the bovine GPR7/8 of the present invention bythe sandwich method according to the present invention, the bovineGPR7/8 monoclonal antibodies of the present invention used for theprimary and the secondary reactions are preferably antibodies, whichbinding sites to the bovine GPR7/8 of the present invention aredifferent from each other. Thus, the antibodies used in the primary andsecondary reactions are those wherein, when the antibody used in thesecondary reaction recognizes the C-terminal region of the bovine GPR7/8of the present invention, the antibody recognizing the site other thanthe C-terminal regions, e.g., recognizing the N-terminal region, ispreferably used in the primary reaction.

The bovine GPR7/8 antibody of the present invention may be used in anassay system other than the sandwich method, such as the competitivemethod, the immunometric method or the nephrometry.

In the competitive method, an antigen in a sample fluid and a labeledantigen are competitively reacted with an antibody, then an unreactedlabeled antigen (F) and a labeled antigen bound to the antibody (B) areseparated (i.e., B/F separation) and the labeled amount of either B or Fis measured to determine the amount of the antigen in the sample fluid.In the reactions for such a method, there are a liquid phase method inwhich a soluble antibody is used as the antibody and the B/F separationis effected by polyethylene glycol, while a second antibody to theantibody is used, and a solid phase method in which an immobilizedantibody is used as the first antibody or a soluble antibody is used asthe first antibody, while an immobilized antibody is used as the secondantibody.

In the immunometric method, an antigen in a sample fluid and animmobilized antigen are competitively reacted with a given amount of alabeled antibody followed by separating the solid phase from the liquidphase; or an antigen in a sample fluid and an excess amount of labeledantibody are reacted, then an immobilized antigen is added to bind anunreacted labeled antibody to the solid phase and the solid phase isseparated from the liquid phase. Thereafter, the labeled amount of anyof the phases is measured to determine the antigen amount in the samplefluid.

In the nephrometry, the amount of insoluble sediment, which is producedas a result of the antigen-antibody reaction in a gel or in a solution,is measured. Even when the amount of an antigen in a sample fluid issmall and only a small amount of the sediment is obtained, a lasernephrometry utilizing laser scattering can be suitably used.

In applying each of those immunoassays to the assay method of thepresent invention, any special conditions, operations, etc. are notrequired. The assay system for the bovine GPR7/8 of the presentinvention may be constructed in addition to conditions or operationsconventionally used for each of the methods, taking technicalconsideration by one skilled in the art into account. For the details ofsuch conventional technical means, a variety of reviews, referencebooks, etc. may be referred to:

for example, Hiroshi Irie (ed.): “Radioimmunoassay” (published byKodansha, 1974); Hiroshi Irie (ed:): “Radioimmunoassay; Second Series”(published by Kodansha, 1979); Eiji Ishikawa, et al. (ed.): “EnzymeImmunoassay” (published by Igaku Shoin, 1978); Eiji Ishikawa, et al.(ed.): “Enzyme lhmunoassay” (Second Edition) (published by Igaku Shoin,1982); Eiji Ishikawa, et al. (ed.): “Enzyme Immunoassay” (Third Edition)(published by Igaku Shoin, 1987); “Methods in Enzymology” Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part. D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (all published by Academic Press); etc.

As described above, the bovine GPR7/8 of the present invention can bequantified with high sensitivity, using the bovine GPR7/8 antibody ofthe present invention.

Furthermore, when a reduced level of the bovine GPR7/8 of the presentinvention is detected by quantifying a level of the bovine GPR7/8 of thepresent invention using, the bovine GPR7/8 antibody of the presentinvention, it can be diagnosed that one suffers from, e.g., anorexia,hypertension, autoimmune disease, heart failure, cataract, glaucoma,acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially, anorexia or the like) etc.; or itis highly likely for one to suffer from these disease in the future.

When an increased level of the bovine GPR7/8 of the present invention isdetected, it can be diagnosed that one suffers from, e.g., obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma, Sheehan's syndrome,spermatogenesis disorder (especially, obesity, or the like), etc.; or itis highly likely for one to suffer from these disease in the future.

The bovine GPR7/8 antibody of the present invention may also be employedto detect the bovine GPR7/8 of the present invention present in a samplefluid such as body fluids, tissues, etc. The antibody may further beused for the preparation of an antibody column to purify the bovineGPR7/8 of the present invention, detect the bovine GPR7/8, of thepresent invention in each fraction upon purification, analysis of thebehavior of the bovine GPR7/8 of the present invention in the cellsunder investigation.

(4) Gene Diagnostic Agent

By using the bovine GPR7/8 DNA of the present invention, e.g., as aprobe, abnormality (gene abnormality) of the DNA or mRNA encoding thebovine GPR7/8 of the present invention in human or other warm-bloodedanimals (e.g., rats, mice, guinea pigs, rabbits, chicken, sheep, swine,bovine, horses, cats, dogs, monkeys, etc.) can be detected. Thus, thebovine GPR7/8 DNA of the present invention is useful as a genediagnostic agent for the damage to the DNA or mRNA, mutation, adecreased expression or an increased expression, or overexpression ofthe DNA or mRNA.

The gene diagnosis described above using the bovine GPR7/8 DNA of thepresent invention can be performed by, for example, the publicly knownnorthern hybridization assay, or the PCR-SSCP assay (Genomics, 5,874-879 (1989); Proceedings of the National Academy of Sciences of theUnited States of America, 86, 2766-2770 (1989)), etc.

When a decreased expression of mRNA is detected, e.g., by northernhybridization, it can be diagnosed that one is likely to suffer from,for example, anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation; arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease (especially anorexia or the like) etc.; or itis highly likely for one to suffer from diseases in the future.

When overexpression of mRNA is detected by northern hybridization, itcan be diagnosed that one is likely to suffer from, for example, obesity(e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like); or itis highly likely for one to suffer from diseases in the future.

(5) Pharmaceutical Composition Comprising Bovine GPR7/8 Antisense DNA

The antisense DNA that binds complementarily to the bovine GPR7/8 DNA ofthe present invention to inhibit expression of the DNA can be used aspreventive/therapeutic agents, for example, for obesity (e.g., malignantmastocytosis, exogenous obesity, hyperinsulinar obesity, hyperplasmicobesity, hypophyseal adiposity, hypoplasmic obesity, hypothyroidobesity, hypothalamic obesity, symptomatic obesity, infantile obesity,upper body obesity, alimentary obesity, hypogonadal obesity, systemicmastocytosis, simple obesity, central obesity, etc.), hyperphagia,pituitary tumor, diencephalon tumor, menstrual disorder, autoimmunedisease, prolactinoma, sterility, impotence, amenorrhea, lactorrhea,acromegaly, Chiari-Frommel syndrome, Argonz-del Castillo syndrome,Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity or the like), etc.

For example, when the antisense DNA is used, the antisense DNA may beadministered directly, or the DNA is inserted into an appropriate vectorsuch as retrovirus vector, adenovirus vector, adenovirus-associatedvirus vector, etc. and then administered in a conventional manner. Theantisense DNA may also be administered as an intact DNA, or preparedinto pharmaceutical preparations together with a physiologicallyacceptable carrier such as an adjuvant to assist its uptake andadministered by gene gun or through a catheter such as a catheter with ahydrogel.

In addition, the antisense DNA may also be employed as anoligonucleotide probe for diagnosis to examine the presence of thebovine GPR7/8 DNA of the present invention in tissues or cells andstates of its expression.

(6) Pharmaceutical Composition Comprising the Bovine GPR7/8 Antibody ofthe Present Invention

The bovine GPR7/8 antibody of the present invention having an effect toneutralize the activity of the bovine GPR7/8 of the present inventioncan be used as drugs for the prevention/treatment of, for example,obesity (e.g., malignant mastocytosis, exogenous obesity, hyperinsulinarobesity, hyperplasmic obesity, hypophyseal adiposity, hypoplasmicobesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity,infantile obesity, upper body obesity, alimentary obesity, hypogonadalobesity, systemic mastocytosis, simple obesity, central obesity, etc.),hyperphagia, pituitary tumor, diencephalon tumor, menstrual disorder,autoimmune disease, prolactinoma, sterility, impotence, amenorrhea,lactorrhea, acromegaly, Chiari-Frommel syndrome, Argonz-del Castillosyndrome, Forbes-Albright syndrome, lymphoma or Sheehan's syndrome,spermatogenesis disorder, etc. (especially, obesity, or the like), etc.

The therapeutic/preventive agent comprising the bovine GPR7/8 antibodyof the present invention can be manufactured in a similar manner to thepharmaceuticals comprising the antibody to the peptide of the presentinvention described above and provided for use.

(7) Bovine GPR7/8 DNA Transgenic Animal

The present invention provides a non-human mammal bearing an exogenousDNA encoding the bovine GPR7/8 of the present invention (hereinaftermerely referred to as the exogenous bovine GPR7/8 DNA of the presentinvention) or its variant DNA (sometimes simply referred to as theexogenous variant bovine GPR7/8 DNA of the present invention).

Thus, the present invention provides:

(i) a non-human mammal bearing the exogenous bovine GPR7/8 DNA or itsvariant DNA;

(ii) the mammal according to (i), wherein the non-human mammal is arodent;

(iii) the mammal according to (ii), wherein the rodent is mouse or rat;and,

(iv) a recombinant vector bearing the exogenous bovine GPR7/8 DNA of thepresent invention or its variant DNA and capable of expressing in amammal.

The bovine GPR7/8 DNA transgenic animals of the present invention can beprepared in a manner similar to the DNA transgenic animals of thepresent invention described above.

In a non-human mammal bearing the normal bovine GPR7/8 DNA of thepresent invention, the normal bovine GPR7/8 DNA of the present inventionis expressed to a high level, and may eventually develop thehyperfunction of the bovine GPR7/8 of the present invention by promotingthe function of endogenous normal bovine GPR7/8 DNA. Therefore, theanimal can be utilized as a pathologic model animal for such a disease.Specifically, using the normal bovine GPR7/8 DNA transgenic animal ofthe present invention, it is possible to elucidate the mechanism of thehyperfunction of the bovine GPR7/8 of the present invention and thepathological mechanism of the disease associated with the bovine GPR7/8of the present invention and to determine how to treat the disease.

Furthermore, since a mammal wherein the exogenous normal bovine GPR7/8DNA of the present invention is transfected exhibits an increasingsymptom of the bovine GPR7/8 of the present invention librated, theanimal is usable for screening therapeutic agents for the diseaseassociated with the bovine GPR7/8 of the present invention.

On the other hand, in a non-human mammal bearing the abnormal bovineGPR7/8 DNA of the present invention, the abnormal bovine GPR7/8 DNA ofthe present invention is expressed at a high level, the animal may bethe function inactivation type in adaptability of the bovine GPR7/8 ofthe present invention by inhibiting the function of the endogenousnormal bovine GPR7/8 DNA and can be utilized as its disease modelanimal. For example, using the abnormal bovine GPR7/8 DNA-transonicanimal of the present invention, it is possible to elucidate themechanism of in adaptability of the bovine GPR7/8 of the presentinvention and to perform to study a method for treatment of thisdisease.

More specifically, the transonic animal of the present inventionexpressing the abnormal bovine GPR7/8 DNA of the present invention to ahigh level is also expected to serve as an experimental model for theelucidation of the mechanism of the functional inhibition (dominantnegative effect) of normal bovine GPR7/8 by the abnormal bovine GPR7/8of the present invention in the function inactive type in adaptabilityof the bovine GPR7/8 of the present invention.

The abnormal exogenous bovine GPR7/8 DNA transonic mammal of the presentinvention is also expected to serve for screening a candidate drug forthe treatment of the function inactive type in adaptability of thebovine GPR7/8 of the present invention, since the bovine GPR7/8 of thepresent invention is increased in such an animal in its free form.

Other potential applications of two kinds of the bovine GPR7/8 DNAtransonic animals described above include:

(1) use as a cell source for tissue culture;

(2) elucidation of the relation to a peptide that is specificallyexpressed or activated by the bovine GPR7/8 of the present invention, bydirect analysis of DNA or RNA in tissues of the bovine GPR7/8 DNAtransonic animal of the present invention or by analysis of a peptidetissue expressed by the DNA;

(3) research in the function of cells derived from tissues that arecultured usually only with difficulty, using cells of tissue bearing theDNA cultured by a standard tissue culture technique;

(4) screening of a drug that enhances the functions of cells using thecells described in (3) above; and,

(5) isolation and purification of the variant bovine GPR7/8 of thepresent invention and preparation of an antibody thereto.

Furthermore, clinical conditions of a disease associated with the bovineGPR7/8 of the present invention, including the function inactive type inadaptability of the bovine GPR7/8 of the present invention can bedetermined using the bovine GPR7/8 DNA transonic animal of the presentinvention. Also, pathological findings on each organ in a disease modelassociated with the bovine GPR7/8 of the present invention can beobtained in more detail, leading to the development of a new method fortreatment as well as the research and therapy of any secondary diseasesassociated with the disease.

It is also possible to obtain a free DNA-transfected cell by withdrawingeach organ from the bovine GPR7/8 DNA transonic animal of the presentinvention, mincing the organ and degrading with a proteinase such astrying, etc., followed by establishing the line of culturing or culturedcells. Furthermore, the transonic animal can serve as identification ofcells capable of producing the bovine GPR7/8 of the present invention,and as studies on association with apropos, differentiation orpropagation or on the mechanism of signal transduction in theseproperties to inspect any abnormality therein. Thus, the transonicanimal can provide an effective research material for the bovine GPR7/8of the present invention and for elucidating the function and effectthereof.

To develop a therapeutic drug for the treatment of diseases associatedwith the bovine GPR7/8 of the present invention, including the functioninactive type in adaptability of the bovine GPR7/8 of the presentinvention, using the bovine GPR7/8 DNA transonic animal of the presentinvention, an effective and rapid method for screening can be providedby using the method for inspection and the method for quantification,etc. Described above. It is also possible to investigate and develop amethod for DNA therapy for the treatment of diseases associated with thebovine GPR7/8 of the present invention, using the bovine GPR7/8 DNAtransonic animal of the present invention or a vector capable ofexpressing the exogenous bovine GPR7/8 DNA of the present invention.

(8) Knockout Animal

The present invention provides a non-human mammal embryonic stem cellbearing the bovine GPR7/8 DNA of the present invention inactivated and anon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention.

Thus, the present invention provides:

(i) a non-human embryonic stem cell in which the bovine GPR7/8 DNA ofthe present invention is inactivated;

(ii) an embryonic stem cell according to (i), wherein the DNA isinactivated by introducing a reporter gene (e.g., β-galactosidase genederived from Escherichia coli);

(iii) an embryonic stem cell according to (i), which is resistant toneomycin;

(iv) an embryonic stem cell according to (i), wherein the non-humanmammal is a rodent;

(v) an embryonic stem cell according to (iv), wherein the rodent ismouse;

(vi) a non-human mammal deficient in expressing the bovine GPR7/8 DNA ofthe present invention, wherein the DNA is inactivated;

(vii) a non-human mammal according to (vi), wherein the DNA isinactivated by inserting a reporter gene (e.g., β-galactosidase derivedfrom Escherichia coli) therein and the reporter gene is capable of beingexpressed under control of a promoter for the DNA of the presentinvention;

(viii) a non-human mammal according to (vi), which is a rodent;

(ix) a non-human mammal according to (viii), wherein the rodent ismouse; and,

(x) a method of screening a compound or its salt that promotes orinhibits the promoter activity for the bovine GPR7/8 DNA of the presentinvention, which comprises administering a test compound to the mammalof (vii) and detecting expression of the reporter gene.

The non-human mammal embryonic stem cell wherein the bovine GPR7/8 DNAof the present invention is inactivated, and the non-human mammaldeficient in expressing the bovine GPR7/8 DNA of the present inventionwherein the DNA is inactivated can be prepared as in the non-humanmammal embryonic stem cell of the present invention and the non-humanmammal deficient in expressing the DNA of the present inventiondescribed above.

The non-human mammal embryonic stem cell, in which the bovine GPR7/8 DNAof the present invention is inactivated, is very useful for preparing anon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention.

Since the non-human mammal deficient in expressing the bovine GPR7/8 DNAof the present invention lacks various biological activities derivedfrom the bovine GPR7/8 of the present invention, such an animal can be adisease model suspected of inactivated biological activities of thebovine GPR7/8 of the present invention and thus, offers an effectivestudy to investigate causes for and therapy for these diseases.

(8a) Method of Screening Compounds having Therapeutic/Preventive Effectson Diseases Caused by Deficiency, Damages, etc. of the Bovine GPR7/8 DNAof the Present Invention

The non-human mammal deficient in expressing the bovine GPR7/8 DNA ofthe present invention can be employed for screening of compounds havingtherapeutic/prophylactic effects on diseases caused by deficiency,damages, etc. of the bovine GPR7/8 DNA of the present invention.

That is, the present invention provides a method for screening of acompound or its salt having therapeutic/preventive effects on diseasescaused by deficiency, damages, etc. of the bovine GPR7/8 DNA of thepresent invention, which comprises administering a test compound to thenon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention and observing/measuring a change occurred in theanimal.

As the non-human mammal deficient in expressing the bovine GPR7/8-DNA ofthe present invention which can be employed for the screening method,the same examples as given hereinabove apply.

Examples of the test compounds include peptides, proteins, non peptidecompounds, synthetic compounds, fermentation products, cell extracts,vegetable extracts, animal tissue extracts, blood plasma, etc. Thesecompounds may be novel compounds or publicly known compounds.

Specifically, the non-human mammal deficient in expressing the bovineGPR7/8 DNA of the present invention is treated with a test compound,comparison is made with an intact animal for control and a change ineach organ, tissue, disease conditions, etc. of the animal is used as anindicator to assess the therapeutic/prophylactic effects of the testcompound.

For treating an animal to be test with a test compound, for example,oral administration, intravenous injection, etc. are applied and thetreatment is appropriately selected depending upon conditions of thetest animal, properties of the test compound, etc. Furthermore, a doseof test compound to be administered can be appropriately chosendepending on method for administration, nature of the test compound,etc.

In screening compounds having the therapeutic/preventive effect on,e.g., anorexia, hypertension, autoimmune disease, heart failure,cataract, glaucoma, acute bacterial meningitis, acute myocardialinfarction, acute pancreatitis, acute viral encephalitis, adultrespiratory distress syndrome, alcoholic hepatitis, Alzheimer's disease,asthma, arteriosclerosis, atopic dermatitis, bacterial pneumonia,bladder cancer, fracture, breast cancer, bulimia, polyphagia, burnhealing, uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis, osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia, or the like), thenon-human mammal deficient in expressing the bovine GPR7/8 DNA of thepresent invention is subjected to a sugar loading treatment, a testcompound is administered before or after the sugar loading treatmentand, blood sugar level, body weight change, etc. of the animal ismeasured with passage of time.

In the screening method described above, when a test compound isadministered to a test animal and found to reduce the blood sugar levelof the animal to at least about 10%, preferably at least about 30% andmore preferably at least about 50%, the test compound can be selected tobe a compound having a therapeutic/preventive effect on the diseasesabove.

The compound obtained using the screening method above is a compoundselected from the test compounds described above and exhibits atherapeutic/preventive effect on the diseases caused by deficiencies,damages, etc. of the bovine GPR7/8 of the present invention. Therefore,the compound can be employed as a safe and low toxic drug for thetreatment and prevention of these diseases. Furthermore, compoundsderived from such a compound obtained by the screening described abovecan be similarly employed.

The compound obtained by the screening method above may be in the formof salts. As such salts, there may be used salts with physiologicallyacceptable acids (e.g., inorganic acids, organic acids, etc.) or bases(e.g., alkali metal salts, etc.), preferably in the form ofphysiologically acceptable acid addition salts. Examples of such saltsare salts with inorganic acids (e.g., hydrochloric acid, phosphoricacid, hydrobromic acid, sulfuric acid, etc.), salts with organic acids(e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleicacid, succinic acid, tartaric acid, citric acid, malic acid, oxalicacid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.)and the like.

A pharmaceutical composition comprising the compound or its salt,obtained by the above screening method, may be manufactured in a mannersimilar to the method for preparing the pharmaceutical compositioncomprising the peptide of the present invention described hereinabove.

Since the pharmaceutical composition thus obtained is safe and lowtoxic, it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

A dose of the compound or its salt to be administered varies dependingupon particular disease, subject to be administered, route ofadministration, etc., and in oral administration to an adult patientwith anorexia (as 60 kg body weight), the compound is administeredgenerally in a dose of approximately 0.1 to. 100 mg, preferablyapproximately 1.0 to 50 mg, more preferably approximately 1.0 to 20 mgper day. For parenteral administration to an adult patient with anorexia(as 60 kg body weight), it is advantageous to administer the compoundintravenously in the form of an injectable preparation in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day, though the single dosagevaries depending upon particular subject, particular disease, etc. Forother animals, the compound can be administered in the correspondingdose with converting it into that for the 60 kg body weight.

(8b) Method of Screening a Compound that Promotes or Inhibits theActivity of a Promoter to the Bovine GPR7/8 DNA of the Present Invention

The present invention provides a method of screening a compound or itssalt that promotes or inhibits the activity of a promoter to the bovineGPR7/8 DNA of the present invention, which comprises administering atest compound to a non-human mammal deficient in expressing the bovineGPR7/8 DNA of the present invention and detecting expression of thereporter gene.

In the screening method described above, the non-human mammal deficientin expressing the bovine GPR7/8 DNA of the present invention is selectedfrom the aforesaid non-human mammal deficient in expressing the bovineGPR7/8 DNA of the present invention, as an animal in which the bovineGPR7/8 DNA of the present invention is inactivated by introducing areporter gene and the reporter gene is expressed under control of apromoter to the bovine GPR7/8 DNA of the present invention.

The same examples of the test compound apply to those given above.

As the reporter gene, the same specific examples apply. Preferablyemployed are β-galactosidase (lacZ), soluble alkaline phosphatase gene,luciferase gene and the like.

Since the reporter gene is present under control of a promoter to thebovine GPR7/8 DNA of the present invention in the non-human mammaldeficient in expressing the bovine GPR7/8 DNA of the present inventionwherein the bovine GPR7/8 DNA of the present invention is substitutedwith the reporter gene, the activity of the promoter can be detected bytracing expression of a substance encoded by the reporter gene.

For example, when a part of the DNA region encoding the bovine GPR7/8 ofthe present invention is substituted with, e.g., β-galactosidase gene(lacZ) derived from Escherichia coli, β-galactosidase is expressed in atissue where the bovine GPR7/8 of the present invention shouldoriginally be expressed, instead of the bovine GPR7/8 of the presentinvention. Thus, the state of expression condition of the bovine GPR7/8of the present invention can be readily observed in vivo of an animal bystaining with a reagent, e.g.,5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) which issubstrate for β-galactosidase. Specifically, a mouse deficient in thebovine GPR7/8 of the present invention, or its tissue slice section isfixed with glutaraldehyde, etc. After washing with phosphate bufferedsaline (PBS), the system is reacted with a staining solution containingX-gal at room temperature or about 37° C. for approximately 30 minutesto an hour. After the β-galactosidase reaction is terminated by washingthe tissue preparation with 1 mM EDTA/PBS solution, the color formed isobserved. Alternatively, mRNA encoding lacZ may be detected in aconventional manner.

The compound or salts thereof obtained using the aforesaid screeningmethod are compounds that are selected from the test compounds describedabove and the compounds that promote or inhibit the activity of apromoter to the bovine GPR7/8 DNA of the present invention.

The compound obtained by the screening method above may form salts. Assalts of the compound, there may be used salts with physiologicallyacceptable acids (e.g., inorganic acids, etc.) or bases (e.g., organicacids, etc.), and especially preferred are physiologically acceptableacid addition salts. Examples of such salts are salts with inorganicacids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid,sulfuric acid, etc.), salts with organic acids (e.g., acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid, etc.) and the like.

The compound or its salt that promotes the promoter activity to thebovine GPR7/8 DNA of the present invention can promote expression of thebovine GPR7/8 of the present invention thereby to promote the functionof the bovine GPR7/8. Thus, these compounds are useful as safe andlow-toxic drugs for the treatment/prevention of diseases, e.g.,anorexia, hypertension, autoimmune disease, heart failure, cataract,glaucoma, acute bacterial meningitis, acute myocardial infarction, acutepancreatitis, acute viral encephalitis, adult respiratory distresssyndrome, alcoholic hepatitis, Alzheimer's disease, asthma,arteriosclerosis, atopic dermatitis, bacterial pneumonia, bladdercancer, fracture, breast cancer, bulimia, polyphagia, burn healing,uterine cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, chronic pancreatitis, liver cirrhosis, cancer ofthe colon and rectum (colon cancer/rectal cancer), Crohn's disease,dementia, diabetic complications, diabetic nephropathy, diabeticneuropathy, diabetic retinopathy, gastritis, Helicobacter pyloribacterial infectious disease, hepatic insufficiency, hepatitis A,hepatitis B, hepatitis C, hepatitis, herpes simplex virus infectiousdisease, varicellazoster virus infectious disease, Hodgkin's disease,AIDS infectious disease, human papilloma virus infectious disease,hypercalcemia, hypercholesterolemia, hyperglyceridemia, hyperlipemia,infectious disease, influenza infectious disease, insulin dependentdiabetes mellitus (type I), invasive staphylococcal infectious disease,malignant melanoma, cancer metastasis, multiple myeloma, allergicrhinitis, nephritis, non-Hodgkin's lymphoma, insulin-independentdiabetes mellitus (type II), non-small cell lung cancer, organtransplantation, arthrosteitis osteomalacia, osteopenia, osteoporosis,ovarian cancer, Behcet's disease of bone, peptic ulcer, peripheralvessel disease, prostatic cancer, reflux esophagitis, renalinsufficiency, rheumatoid arthritis, schizophrenia, sepsis, septicshock, severe systemic fungal infectious disease, small cell lungcancer, spinal injury, stomach cancer, systemic lupus erythematosus,transient cerebral ischemia, tuberculosis, cardiac valve failure,vascular/multiple infarction dementia, wound healing, insomnia,arthritis, pituitary hormone secretion disorders [e.g., prolactinsecretion disorders (e.g., hypoovarianism, spermatic underdevelopment,menopausal symptoms, hypothyroidism, etc.)], pollakiuria, uremia,neurodegenerative disease, etc. (especially, anorexia or the like) orthe like (especially, appetite (eating) stimulant).

The compound or its salt that inhibits the activity of a promoter to thebovine GPR7/8 DNA of the present invention can inhibit expression of thebovine GPR7/8 of the present invention thereby to inhibit the functionof the bovine GPR7/8. Thus, these compounds are useful as drugs,including preventive/therapeutic drugs (prolactin production inhibitors)for diseases, for example, obesity (e.g., malignant mastocytosis,exogenous obesity, hyperinsulinar obesity, hyperplasmic obesity,hypophyseal adiposity, hypoplasmic obesity, hypothyroid obesity,hypothalamic obesity, symptomatic obesity, infantile obesity, upper bodyobesity, alimentary obesity, hypogonadal obesity, systemic mastocytosis,simple obesity, central obesity, etc.), hyperphagia, pituitary tumor,diencephalon tumor, menstrual disorder, autoimmune disease,prolactinoma, sterility, impotence, amenorrhea, lactorrhea, acromegaly,Chiari-Frommel syndrome, Argonz-del Castillo syndrome, Forbes-Albrightsyndrome, lymphoma or Sheehan's syndrome, spermatogenesis disorders,etc.; preferably as preventive/therapeutic agents for obesity,hyperphagia, etc.

Furthermore, compounds derived from the compounds obtained by thescreening described above may be likewise used.

The pharmaceuticals comprising the compound or its salt obtained by thescreening method may be manufactured as in the aforesaid pharmaceuticalscomprising the peptide of the present invention or its salt.

Since the pharmaceutical preparation thus obtained is safe and lowtoxic; it can be administered to human or mammals (e.g., rats, mice,guinea pigs, rabbits, sheep, swine, bovine, horses, cats, dogs, monkeys,etc.).

A dose of the compound or its salt to be administered varies dependingupon target disease, subject to be administered, route ofadministration, etc.; when the compound that promotes the promoteractivity to the bovine GPR7/8 DNA of the present invention is orallyadministered to an adult patient, e.g., with anorexia (as 60 kg bodyweight), the compound is administered generally in a dose ofapproximately 0.1 to 100 mg, preferably approximately 1.0 to 50 mg, morepreferably approximately 1.0 to 20 mg per day. In parenteraladministration, a single dose of the compound varies depending uponsubject to be administered, target disease, etc. When the compound thatpromotes the promoter activity to the bovine GPR7/8 DNA of the presentinvention is administered to an adult patient with, e.g., anorexia (as60 kg body weight) in the form of an injectable preparation, it isadvantageous to administer the compound intravenously in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day. For other animals, thecompound can be administered in the corresponding dose with convertingit into that for the 60 kg body weight.

On the other hand, when a compound that inhibits the promoter activityto the bovine GPR7/8 DNA of the present invention is orallyadministered, the compound is orally administered to an adult patientwith obesity (as 60 kg body weight) generally in a dose of approximately0.1 to 100 mg, preferably approximately 1.0 to 50 mg, more preferablyapproximately 1.0 to 20 mg per day. In parenteral administration, asingle dose of the compound varies depending upon subject to beadministered, target disease, etc. For example, when the compound thatinhibits the promoter activity to the bovine GPR7/8 DNA of the presentinvention is administered to an adult patient with obesity (as 60 kgbody weight) in the form of an injectable preparation, it isadvantageous to administer the compound intravenously in a dose ofapproximately 0.01 to 30 mg, preferably approximately 0.1 to 20 mg, morepreferably approximately 0.1 to 10 mg per day. For other animals, thecompound can be administered in the corresponding dose with convertingit into that for the 60 kg body weight.

As described above, the non-human mammal deficient in expressing thebovine GPR7/8 DNA of the present invention is extremely useful forscreening a compound or its salt that promotes or inhibits the activityof a promoter to the bovine GPR7/8 DNA of the present invention, and canthus greatly contribute to investigations of causes for various diseasescaused by failure to express the bovine GPR7/8 DNA of the presentinvention or to development of preventive/therapeutic agents for thesediseases.

Moreover, when a so-called transonic animal (gene-transfected animal) isprepared by using the bovine GPR7/8 DNA containing the promoter regionof the bovine GPR7/8 of the present invention, ligating genes encodingvarious proteins downstream the same and injecting the genes into animaloocyte, the peptide can be specifically synthesized by the animal sothat it becomes possible to investigate the activity in vivo.Furthermore, when an appropriate reporter gene is ligated to thepromoter region described above to establish a cell line so as toexpress the gene, such can be used as a survey system of low molecularweight compounds that specifically promotes or suppresses the ability ofproducing the bovine GPR7/8 itself of the present invention in vivo.

In the specification and drawings, the codes of bases and amino acidsare shown by abbreviations and in this case, they are denoted inaccordance with the IUPAC-IUB Commission on Biochemical Nomenclature orby the common codes in the art, examples of which are shown below. Foramino acids that may have the optical isomer, L form is presented unlessotherwise indicated.

DNA deoxyribonucleic acid cDNA complementary deoxyribonucleic acid Aadenine T thymine G guanine C cytosine I inosine R adenine (A) orguanine (G) Y thymine (T) or cytosine (C) M adenine (A) or cytosine (C)K guanine (G) or thymine (T) S guanine (G) or cytosine (C) W adenine (A)or thymine (T) B guanine (G), guanine (G) or thymine (T) D adenine (A),guanine (G) or thymine (T) V adenine (A), guanine (G) or cytosine (C) Nadenine (A), guanine (G), cytosine (C) or thymine (T), or unknown orother base RNA ribonucleic acid mRNA messenger ribonucleic acid dATPdeoxyadenosine triphosphate dTTP deoxythymidine triphosphate dGTPdeoxyguanosine triphosphate dCTP deoxycytidine triphosphate ATPadenosine triphosphate EDTA ethylenediaminetetraacetic acid SDS sodiumdodecyl sulfate BHA benzhydrylamine pMBHA p-methyobenzhydrylamine Tosp-toluenesulfonyl Bzl benzyl Bom benzyloxymethyl Boc t-butyloxycarbonylDCM dichloromethane HOBt 1-hydroxybenztriazole DCCN,N′-dicyclohexylcarbodiimide TFA trifluoroacetic acid DIEAdiisopropylethylamine Gly or G glycine Ala or A alanine Val or V valineLeu or L leucine Ile or I isoleucine Ser or S serine Thr or T threonineCys or C cysteine Met or M methionine Glu or E glutamic acid Asp or Daspartic acid Lys or K lysine Arg or R arginine His or H histidine Pheor F phenylalanine Tyr or Y tyrosine Trp or W tryptophan Pro or Pproline Asn or N asparagine Gln or Q glutamine pGlu pyroglutamic acidTyr (I) 3-iodotyrosine DMF N,N-dimethylformamide FmocN-9-fluorenylmethoxycarbonyl Trt trityl Pbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Clt 2-chlorotritylBu^(t) t-butyl Met (O) methionine sulfoxide

The sequence identification numbers in the sequence listing of thespecification indicates the following sequences, respectively.

[SEQ ID NO:1]

This shows the amino acid sequence of human GPR7 ligand A.

[SEQ ID NO:2]

This shows the amino acid sequence of mouse GPR7 ligand A.

[SEQ ID NO:3]

This shows the amino acid sequence of rat GPR7 ligand A.

[SEQ ID NO:4]

This shows the amino acid sequence of human GPR7 ligand B.

[SEQ ID NO:5]

This shows the amino acid sequence of mouse GPR7 ligand B.

[SEQ ID NO:6]

This shows the amino acid sequence of rat GPR7 ligand B.

[SEQ ID NO:7]

This shows the amino acid sequence of human GPR7 ligand C.

[SEQ ID NO:8]

This shows the amino acid sequence of human GPR7 ligand D.

[SEQ ID NO:9]

This shows the amino acid sequence of mouse GPR7 ligand C.

[SEQ ID NO:10]

This shows the amino acid sequence of mouse GPR7 ligand D.

[SEQ ID NO:11]

This shows the amino acid sequence of rat GPR7 ligand C.

[SEQ ID NO:12]

This shows the amino acid sequence of rat GPR7 ligand D.

[SEQ ID NO:13]

This shows the amino acid sequence of human GPR7 ligand E.

[SEQ ID NO:14]

This shows the amino acid sequence of mouse GPR7 ligand E.

[SEQ ID NO:15]

This shows the amino acid sequence of rat GPR7 ligand E.

[SEQ ID NO:16]

This shows the amino acid sequence of human GPR7 ligand F.

[SEQ ID NO:17]

This shows the amino acid sequence of mouse GPR7 ligand F.

[SEQ ID NO:18]

This shows the amino acid sequence of rat GPR7 ligand F.

[SEQ ID NO:19]

This shows the amino acid sequence of human GPR7 ligand precursor Gcontaining no secretory signal.

[SEQ ID NO:20]

This shows the amino acid sequence of mouse GPR7 ligand precursor Gcontaining no secretory signal.

[SEQ ID NO:21]

This shows the amino acid sequence of rat GPR7 ligand precursor Gcontaining no secretory signal.

[SEQ ID NO:22]

This shows the amino acid sequence of human GPR7 ligand precursor Hcontaining a secretory signal.

[SEQ ID NO:23]

This shows the amino acid sequence of mouse GPR7 ligand precursor Hcontaining a secretory signal.

[SEQ ID NO:24]

This shows the amino acid sequence of rat GPR7 ligand precursor Hcontaining a secretory signal.

[SEQ ID NO:25]

This shows the base sequence of DNA encoding human GPR7 ligand A.

[SEQ ID NO:26]

This shows the base sequence of DNA encoding mouse GPR7 ligand A.

[SEQ ID NO:27]

This shows the base sequence of DNA encoding rat GPR7 ligand A.

[SEQ ID NO:28]

This shows the base sequence of DNA encoding human GPR7 ligand B.

[SEQ ID NO:29]

This shows the base sequence of DNA encoding mouse GPR7 ligand B.

[SEQ ID NO:30]

This shows the base sequence of DNA encoding rat GPR7 ligand B.

[SEQ ID NO:31]

This shows the base sequence of DNA encoding human GPR7 ligand C.

[SEQ ID NO:32]

This shows the base sequence of DNA encoding human GPR7 ligand D.

[SEQ ID NO:33]

This shows the base sequence of DNA encoding mouse GPR7 ligand C.

[SEQ ID NO:34]

This shows the base sequence of DNA encoding mouse GPR7 ligand D.

[SEQ ID NO:35]

This shows the base sequence of DNA encoding rat GPR7 ligand C.

[SEQ ID NO:36]

This shows the base sequence of DNA encoding rat GPR7 ligand D.

[SEQ ID NO:37]

This shows the base sequence of DNA encoding human GPR7 ligand E.

[SEQ ID NO:38]

This shows the base sequence of DNA encoding mouse GPR7 ligand E.

[SEQ ID NO:39]

This shows the base sequence of DNA encoding rat GPR7 ligand E.

[SEQ ID NO:40]

This shows the base sequence of DNA encoding human GPR7 ligand F.

[SEQ ID NO:41]

This shows the base sequence of DNA encoding mouse GPR7 ligand F.

[SEQ ID NO:42]

This shows the base sequence of DNA encoding rat GPR7 ligand F.

[SEQ ID NO:43]

This shows the base sequence of DNA encoding human GPR7 ligand precursorG containing no secretory signal.

[SEQ ID NO:44]

This shows the base sequence of DNA encoding mouse GPR7 ligand precursorG containing no secretory signal.

[SEQ ID NO:45]

This shows the base sequence of DNA encoding rat GPR1 ligand precursor Gcontaining no secretory signal.

[SEQ ID NO:46]

This shows the base sequence of DNA encoding human GPR7 ligand precursorH containing a secretory signal.

[SEQ ID NO:47]

This shows the base sequence of DNA encoding mouse GPR7 ligand precursorH containing a secretory signal.

[SEQ ID NO:48]

This shows the base sequence of DNA encoding rat GPR7 ligand precursor Hcontaining a secretory signal.

[SEQ ID NO:49]

This shows the amino acid sequence of human GPR7.

[SEQ ID NO:50]

This shows the base sequence of a DNA containing the DNA encoding humanGPR7.

[SEQ ID NO:51]

This shows a synthetic DNA used in EXAMPLE 1 to screen cDNA encodinghuman GPR7 ligand precursor H.

[SEQ ID NO:52]

This shows a synthetic DNA used in EXAMPLE 1 to screen cDNA encodinghuman GPR7 ligand precursor H.

[SEQ ID NO:53]

This shows a synthetic DNA used in EXAMPLE 2 to screen cDNA encodingmouse GPR7 ligand precursor H.

[SEQ ID NO:54]

This shows a synthetic DNA used in EXAMPLE 2 to screen cDNA encodingmouse GPR7 ligand precursor H.

[SEQ ID NO:55]

This shows a synthetic DNA used in EXAMPLE. 3 to screen cDNA encodingrat GPR7 ligand precursor H.

[SEQ ID NO:56]

This shows a synthetic DNA used in EXAMPLE 3 to screen cDNA encoding ratGPR7 ligand precursor H.

[SEQ ID NO:57]

This shows the base sequence of a primer used in REFERENCE EXAMPLE 1.

[SEQ ID NO:58]

This shows the base sequence of a primer used in REFERENCE EXAMPLE 1.

[SEQ ID NO:59]

This shows the amino acid sequence of rat TGR26.

[SEQ ID NO:60]

This shows the base sequence of DNA encoding rat TGR26.

[SEQ ID NO:61]

This shows the base sequence of Primer 1 used for PCR in REFERENCEEXAMPLE 3.

[SEQ ID NO:62]

This shows the base sequence of Primer 2 used for PCR in REFERENCEEXAMPLE 3.

[SEQ ID NO:63]

This shows the base sequence of a primer used in EXAMPLE 9.

[SEQ ID NO:64]

This shows the base sequence of a primer used in EXAMPLE 9.

[SEQ ID NO:65]

This shows the base sequence of a primer used in EXAMPLE 9.

[SEQ ID NO:66]

This shows the amino acid sequence of bovine GPR7 ligand A.

[SEQ ID NO:67]

This shows the amino acid sequence of bovine GPR7 ligand B.

[SEQ ID NO:68]

This shows the amino acid sequence of bovine GPR7 ligand C.

[SEQ ID NO:69]

This shows the amino acid sequence of bovine GPR7 ligand D.

[SEQ ID NO:70]

This shows the amino acid sequence of bovine GPR7 ligand E.

[SEQ ID NO:71]

This shows the amino acid sequence of bovine GPR7 ligand F.

[SEQ ID NO:72]

This shows the amino acid sequence of bovine GPR7 ligand precursor Gcontaining no secretory signal.

[SEQ ID NO:73]

This shows the amino acid sequence of bovine GPR7 ligand precursor Hcontaining a secretory signal.

[SEQ ID NO:74]

This shows the base sequence of DNA encoding bovine GPR7 ligand A.

[SEQ ID NO:75]

This shows the base sequence of DNA encoding bovine GPR7 ligand B.

[SEQ ID NO:76]

This shows the base sequence of DNA encoding bovine GPR7 ligand C.

[SEQ ID NO:77]

This shows the base sequence of DNA encoding bovine GPR7 ligand D.

[SEQ ID NO:78]

This shows the base sequence of DNA encoding bovine GPR7 ligand E.

[SEQ ID NO:79]

This shows the base sequence of DNA encoding bovine GPR7 ligand F.

[SEQ ID NO:80]

This shows the base sequence of DNA encoding bovine GPR7 ligandprecursor G containing no secretory signal.

[SEQ ID NO:81]

This shows the base sequence of DNA encoding bovine GPR7 ligandprecursor H containing a secretory signal.

[SEQ ID NO:82]

This shows the base sequence of a primer used in EXAMPLE 12.

[SEQ ID NO:83]

This shows the base sequence of a primer used in EXAMPLE 12.

[SEQ ID NO:84]

This shows the amino acid sequence of human GPR8.

[SEQ ID NO:85]

This shows the base sequence of a DNA containing the DNA encoding humanGPR8.

[SEQ ID NO:86]

This shows the amino acid sequence of bovine GPR7.

[SEQ ID NO:87]

This shows the base sequence of a DNA containing the DNA encoding bovineGPR7.

[SEQ ID NO:88]

This shows the amino acid sequence of bovine GPR8.

[SEQ ID NO:89]

This shows the base sequence of a DNA containing the DNA encoding bovineGPR8.

[SEQ ID NO:90]

This shows the base sequence of a primer used in EXAMPLE 16.

[SEQ ID NO:91]

This shows the base sequence of a primer used in EXAMPLE 16.

[SEQ ID NO:92]

This shows the base sequence of a primer used in EXAMPLE 16.

[SEQ ID NO:93]

This shows the base sequence of a primer used in EXAMPLE 17.

[SEQ ID NO:94]

This shows the base sequence of a primer used in EXAMPLE 17.

[SEQ ID NO:95]

This shows the base sequence of a primer used in EXAMPLE 17.

[SEQ ID NO:96]

This shows the base sequence of a primer used in EXAMPLE 18.

[SEQ ID NO:97]

This shows the base sequence of a primer used in EXAMPLE 18.

[SEQ ID NO:98]

This shows the base sequence of a primer used in EXAMPLE 19.

[SEQ ID NO:99]

This shows the base sequence of a primer used in EXAMPLE 19.

[SEQ ID NO:100]

This shows the amino acid sequence of human GPR8 ligand (1-23).

[SEQ ID NO:101]

This shows the complete DNA sequence, including stop codon, from FIG. 1.

[SEQ ID NO:102]

This shows the complete DNA sequence, including stop codon, from FIG. 3.

[SEQ ID NO:103]

This shows the complete DNA sequence, including stop codon, from FIG. 5.

[SEQ ID NO:104]

This shows the complete DNA sequence, including stop codon, from FIG.20.

[SEQ ID NO:105]

This shows the complete DNA sequence, including stop codon, from FIG.26.

[SEQ ID NO:106]

This shows the complete DNA sequence, including stop codon, from FIG.28.

[SEQ ID NO:107]

This shows an embodiment of SEQ ID NO:4, wherein the N-terminal Trp isof a brominated D-form.

[SEQ ID NO:108]

This shows an embodiment of SEQ ID NO:4, wherein the N-terminal Trp isof a brominated D-form.

Transformant Escherichia ccli JM109/pTAhGPR7-1, which was obtained inEXAMPLE 1 later described, has been deposited as JM109/pTAhGPR7L-1 sinceJun. 27, 2001 on the National Institute of Advanced Industrial Scienceand Technology, International Patent Organism Depositary, located atCentral 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (postal code305-8566), under the Accession Number FERM BP-7640.

Transformant Escherichia coli JM109/pTAmGPR7-1, which was obtained inEXAMPLE 2 later described, has been deposited as JM109/pTAmGPR7L-1 sinceJun. 27, 2001 on the National Institute of Advanced Industrial Scienceand Technology, International Patent Organism Depositary under theAccession Number FERM BP-7641, and since on Jun. 19, 2001 on theInstitute for Fermentation (IFO) under the Accession Number IFO 16656,respectively.

Transformant Escherichia coli JM109/pTArGPR7-1, which was obtained inEXAMPLE 3 later described, has been deposited as JM109/pTArGPR7L-1 sinceJun. 27, 2001 on the National Institute of Advanced Industrial Scienceand Technology, International Patent Organism Depositary under theAccession Number FERM BP-7642, and since on Jun. 19, 2001 on theInstitute for Fermentation (IFO) under the Accession Number IFO 16657,respectively.

Transformant Escherichia coli JM109/pTAbGPR7L-1, which was obtained inEXAMPLE 12 later described, has been deposited since Dec. 17, 2001 onthe National Institute of Advanced. Industrial Science and Technology,International Patent Organism Depositary under the Accession Number FERMBP-7829, and since on Dec. 6, 2001 on the Institute for Fermentation(IFO) under the Accession Number IFO. 16736, respectively.

Transformant Escherichia coli JM109/pTAbGPR7, which was obtained inEXAMPLE 18 later described, has been deposited since May 24, 2002 on theNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary under the Accession Number FERMBP-8050.

Transformant Escherichia coli JM109/pTAbGPR8, which was obtained inEXAMPLE 19 later described, has been deposited since May 24, 2002 on theNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary under the Accession Number FERMBP-8051.

Transformant Escherichia coli DH10B/pAK-rGPR7, which was obtained inREFERENCE EXAMPLE 3 later described, has been deposited since Oct. 31,2000 on the Institute for Fermentation (IFO), located at 2-17-85, JusoHoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan, under the Accession NumberIFO 16496 and since on Nov. 13, 2000 on the Ministry of InternationalTrade and Industry, Agency of Industrial Science and Technology,National Institute of Bioscience and Human Technology (NIBH), located at1-1-3 Higashi, Tsukuba, Ibaraki, Japan, under the Accession Number FERMBP-7365, respectively.

EXAMPLES

The present invention will be described in more detail below, withreference to REFERENCE EXAMPLES and EXAMPLES, but is not deemed to limitthe scope of the present invention thereto.

Reference Example 1

Amplification of Human GPR7 DNA by PCR using Human Chromosomal DNA

By use of human chromosomal DNA as a template, amplification of DNA wascarried out by PCR using two synthetic primers (SEQ ID NO:57 and SEQ IDNO:58). The synthetic primers were constructed so as to amplify the genein the region to be translated to its receptor protein was amplified. Inthis case, the recognition sequences for restriction enzymes were addedat the 5′ and 3′ ends, respectively, so that the base sequencesrecognized by restriction enzymes ClaI and SpeI were added to the geneat the 5′ and 3′ ends, respectively. The reaction solution was composedof 0.5 μl of human chromosomal DNA (TaKaRa Shuzo Co., Ltd.), 1 μM eachof the synthetic DNA primers, 0.8 mM dNTPs, 1 mM MgCl₂ and 1 μl of KODpolymerase (Toyobo Co., Ltd.), to which the buffer attached to theenzymes was added to make the total reaction volume of 50 μl. Foramplification, after heating at 94° C. for 60 seconds, the cycle set toinclude 98° C. for 15 seconds, 65° C. for 2 seconds and 72° C. for 30seconds was repeated 35 times, using Thermal Cycler (TaKaRa Shuzo Co.,Ltd.). The amplified product was confirmed by 0.8% agarose gelelectrophoresis followed by staining with ethidium bromide.

Reference Example 2

Subcloning of the PCR Product into a Plasmid Vector and Confirmation ofthe Amplified DNA Sequence by Decoding the Base Sequence of the InsertedDNA Part

The reaction solution of PCR performed in REFERENCE EXAMPLE 1 wasseparated by 0.8% low melting point agarose gel electrophoresis. Theband part was excised with a razor blade, ground into small pieces,extracted with phenol and then with phenol/chloroform and precipitatedin ethanol to recover DNAs. According to the protocol attached toPCR-Script™ Amp SK(+) Cloning Kit (Stratagene Co.), the recovered DNAswere subcloned to plasmid vector pCR-Script Amp SK(+). The recombinantvector was introduced into Escherichia coli DH5α competent cells (ToyoboCo., Ltd.) to produce transformants. The clones bearing the DNA-insertedfragment were selected in an LB agar medium supplemented withampicillin, IPTG and X-gal. Only the clones exhibiting white color werepicked up with a sterilized toothpick to obtain transformant E. coliDH5α/GPR7. The individual clones were cultured overnight in an LB mediumcontaining ampicillin. Plasmid DNAs were prepared using QIAwell 8Plasmid Kit (QIAGEN, Inc.). An aliquot of the DNAs thus prepared wascleaved by restriction enzymes ClaI and SpeI to confirm the size of thereceptor cDNA fragment inserted. The reaction for base sequencing wascarried out by using a DyeDeoxy Terminator Cycle Sequence Kit (AppliedBiosystems, Inc.), followed by decoding with a fluorescent automaticsequencer (SEQ ID NO:50). The pCR-Script Amp SK(+) plasmid bearing theDNA having the base sequence represented by SEQ ID NO:50 was namedpCR-Script human GPR7. The amino acid sequence of human GPR7 encoded bythe DNA having the base sequence represented by SEQ ID NO:50 ispresented in SEQ ID NO:49. The DNA sequence of human GPR7 sequencedherein differed by 2 bases from the DNA sequence reported by O'Dowd etal. (O'Dowd, B. F. et al., Genomics, 28, 84-91, 1995). These basescorresponding to 893rd and 894th bases in SEQ ID NO:50 were reportedly Cand G in the report by O'Dowd et al., whereas they were found to be Gand C in this REFERENCE EXAMPLE. Based on the findings, the 296th aminoacid of SEQ ID NO:49 in the translated amino acid sequence is Ser inthis EXAMPLE, which was reportedly Thr in O'Dowd et al.

Reference Example 3

Cloning of cDNA Encoding Rat Whole Brain-Derived G Protein-CoupledReceptor Protein and Base Sequencing

By use of rat whole brain cDNA (CLONTECH Labs. Inc.), PCR was carriedout using two primers, Primer 1 (SEQ ID NO:61) and Primer 2 (SEQ IDNO:62) designed from the base sequence of DNA encoding human GPR8. Thereaction solution for PCR wherein 1/10 volume of the above cDNA was usedas a template, was composed of 1/50 volume of Advantage-2 cDNAPolymerase Mix (CLONTECH Labs. Inc.), 0.2 μM of Primer 3, 0.2 μM ofPrimer 2 and 200 μM of dNTPs, to which the buffer attached to theenzymes was added to make the total volume of 25 μl. PCR was carriedout, (1) after heating at 94° C. for 2 minutes, by repeating (2) thecycle set to include 94° C. for 20 seconds and 72° C. for 2 minutes 3times, (3) the cycle set to include 94° C. for 20 seconds, 66° C. for 20seconds and 68° C. for 2 minutes 3 times and (4) the cycle set toinclude 94° C. for 20 seconds, 60° C. for 20 seconds and 68° C. for 2minutes 36 times, finally followed by extension at 68° C. for 7 minutes.The reaction product after the PCR was subcloned to pCR2.1-TOPO(Invitrogen, Inc.) in accordance with the protocol of TA Cloning Kitvector using TA cloning kit (Invitrogen, Inc.). After introducing intoEscherichia coli DH5α, clones bearing cDNA were selected in LB agarmedium supplemented with ampicillin. Individual clones were sequenced toacquire the base sequence (SEQ ID NO:60) of cDNA encoding novel Gprotein-coupled receptor protein. The novel G protein-coupled receptorprotein containing the amino acid sequence (SEQ ID NO:59) encoded by thebase sequence of this DNA was named TGR26.

The amino acid sequence represented by SEQ ID NO:59 had 84.8% homologyto GPR7 (Genomics, 28, 84-91, 1995), which is known human Gprotein-coupled receptor protein.

From the above transformants bearing the plasmid inserted with theTGR26-encoding DNA, one clone was selected and shake cultured in LBmedium supplemented with ampicillin to acquire plasmid. The plasmid wastreated with restriction enzymes ClaI and SpeI to excise the insertencoding TGR26. Using pAKKO-1.11H, which was similarly treated withrestriction enzymes ClaI and SpeI, and Ligation Express Kit (CLONTECH,Inc.), ligation was performed and the ligated product was transfected toEscherichia coli DH10B by electroporation. With respect to the clonethus obtained, the structure of the plasmid for expression cellconstruction was confirmed by the treatment with restriction enzymes andsequencing. The clone was then named Escherichia coli DH10B/pAK-rGPR7.

Reference Example 4

Preparation of TGR26-Expressing CHO Cells

After Escherichia coli DH5α (Toyobo Co., Ltd.) transformed by theexpression plasmid pAK-rGPR7 described in REFERENCE EXAMPLE 3 wascultured, pAK-rGPR7 plasmid DNA was prepared. The plasmid DNA wastransfected to CHO dhfr⁻ cells by using CellPhect Transfection Kit(Amersham Pharmacia Biotech), according to the protocol attached. Aco-precipitated suspension of 5 μg of DNA and calcium phosphate wasadded to 2 Petri dishes of 6 cm diameter, on which 3×10⁵ CHO dhfr⁻ cellshad been plated 48 hours before. After cultivation for a day in MEM amedium containing 10% fetal cow serum, the cells were passaged andcultured in nucleic acid-free MEM a medium containing 10% dialysis fetalcow serum as a selection medium. From the colony of the transformantwhich was TGR26-expressing CHO cells grown in the selection medium, 44clones were selected.

Example 1

Acquisition of GPR7 Ligand Precursor Gene from Human Whole Brain cDNA byPCR and Construction of Expression Plasmid

Using human whole brain cDNA purchased from CLONTECH as a template,amplification was performed by PCR using the following two syntheticDNAs.

(SEQ ID NO:51) GSF1: 5′-GTCGACATGGCCCGGTCCGCGACACTGGCGGCC-3′ (SEQ IDNO:52) GSR2: 5′-GCTAGCAGCGGTGCCAGGAGAGGTCCGGGCTCA-3′

The reaction solution for PCR contained 1 μl of cDNA solution, 0.5 μl ofGSF1 (10 μM), 0.5 μl of GSR2 (10 μM), 2.5 μl of 10× reaction solutionattached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq (CLONTECH, Inc.),to which 17.5 μl of Otsuka distilled water was added to make 25 μl intotal. The reaction solution was applied to PCR using Thermal Cycler9600. The conditions for PCR were set forth: after denaturation at 95°C. for 2 minutes, the cycle set to include 98° C. for 10 seconds, 60° C.for 20 seconds and 72° C. for 20 seconds was repeated 35 times. After itwas confirmed by electrophoresis using an aliquot of the PCR productthat the PCR product of about 400 bp was amplified, the PCR product waspurified using Quiagen PCR purification Kit and directly sequenced toobtain the sequence shown by FIG. 1. The amino acid sequence deducedfrom the DNA sequence of FIG. 1 was the sequence shown in FIG. 2. Next,the PCR product recovered from the gel was subcloned to Escherichia coliJM109 using TA Cloning Kit (Invitrogen, Inc.), to acquire Escherichiacoli JM109/pTAhGPR7-1. Plasmid pTAhGPR7-1 was extracted from Escherichiacoli obtained by the subcloning, using a plasmid extractor (Kurabo Co.,Ltd.) to identify the base sequence of the inserted fragment. It wasconfirmed that the sequence was the same as that of human GPR7 ligandcDNA shown in FIG. 1. Next, after digestion with restriction enzymesSalI and NheI, human GPR7 ligand cDNA fragment of about 0.4 kb wasobtained from the plasmid. Furthermore, expression vector pAKKO-111H foranimal cells was digested by restriction enzymes sites SalI and NheI ofthe multi-cloning sites, and electrophoresed to recover the vectorportion. The human GPR7 ligand cDNA fragment prepared by the foregoingprocedures was ligated through ligation and Escherichia coli JM109 wastransformed to obtain Escherichia coli JM109 to acquire E. coliJM109/pAK-S64.

Transformant E. coli JM109/pAK-S64 was cultured to prepare the DNA ofplasmid pAK-S64 in large quantities.

Example 2

Acquisition of GPR7 Ligand Precursor Gene from Mouse Whole Brain cDNA byPCR

Using mouse whole brain cDNA as a template, amplification was performedby PCR using the following two synthetic DNAs.

(SEQ ID NO:53) MFSAL1: 5′-GTCGACAGCTCCATGGCCCGGTGTAGGACGCTG-3′ (SEQ IDNO:54) MRNHE1: 5′-GCTAGCTCAGGTGCTCTGGCAATCAGTCTCGTG-3′

The reaction solution for PCR contained 1 μl of cDNA solution, 0.5 μl ofMFSAL1 (10 μM), 0.5 μl of MRNHE1 (10 μM), 2.5 μl of 10× reactionsolution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make 25 μl in total. The reaction solution was applied to PCR usingThermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 20 seconds wasrepeated 35 times. After it was confirmed by electrophoresis using analiquot of the PCR product that the PCR product of about 400 bp wasamplified, the PCR product was purified using Quiagen PCR purificationKit and directly sequenced to obtain the sequence shown by FIG. 3. Theamino acid sequence deduced from the DNA sequence of FIG. 3 was thesequence shown in FIG. 4. Next, the PCR product recovered from the gelwas subcloned to Escherichia coli JM109 using TA Cloning Kit(Invitrogen, Inc.) to acquire Escherichia coli JM109/pTAmGPR7-1. PlasmidpTAmGPR7-1 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of mouse GPR7 ligand cDNA shown in FIG. 3.

Example 3

Acquisition of GPR7 Ligand Precursor Gene from Rat Whole Brain cDNA byPCR

Using rat whole brain cDNA as a template, amplification was performed byPCR using the following two synthetic DNAs.

(SEQ ID NO:55) RF: 5′-CACGGCTCCATGGTCCGGTGTAGGACG-3′ (SEQ ID NO:56) RR:5′-CAGCGTCGAGGTTTGGGTTGGGGTTCA-3′

The reaction solution for PCR contained 1 μl of cDNA solution, 0.5 μl ofRF (10 μM), 0.5 μl of RR (10 μM), 2.5 μl of 10× reaction solutionattached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq (CLONTECH, Inc.),to which 17.5 μl of Otsuka distilled water was added to make 25 μl intotal. The reaction solution was applied to PCR using Thermal Cycler9600. The conditions for PCR were set forth: after denaturation at 95°C. for 2 minutes, the cycle set to include 98° C. for 10 seconds, 60° C.for 20 seconds and 72° C. for 20 seconds was repeated 35 times. After itwas confirmed by electrophoresis using an aliquot of the PCR productthat the PCR product of about 400 bp was amplified, the PCR product waspurified using Quiagen PCR purification Kit and directly sequenced toobtain the sequence shown by FIG. 5. The amino acid sequence deducedfrom the DNA sequence of FIG. 5 was the sequence shown in FIG. 6. Next,the PCR product recovered from the gel was subcloned to Escherichia coliJM109 using TA Cloning Kit (Invitrogen, Inc.) to acquire Escherichiacoli JM109/pTArGPR7-1. Plasmid pTArGPR7-1 was extracted from Escherichiacoli obtained by the subcloning, using a plasmid extractor (Kurabo Co.,Ltd.) to identify the base sequence of the inserted fragment. It wasconfirmed that the sequence was the same as that of mouse GPR7 ligandcDNA shown in FIG. 5.

Example 4

Transient Expression of GPR7 Expression Plasmid and Reporter Plasmid inChinese Hamster Ovary (CHO) Cells

Escherichia coli JM109 was transformed using a plasmid prepared byinserting human GPR7 DNA obtained in REFERENCE EXAMPLE 2 into expressionplasmid pAKKO-111H for animal cells by a publicly known method. Afterthe colony obtained was isolated and cultured, GPR7 expression plasmidDNA was prepared using QUIAGEN Plasmid Maxi Kit (QIAGEN, Inc.). Also,plasmid DNA of pCRE-Luc (CLONTECH, Inc.), in which luciferase gene wasligated as a reporter at the downstream of cAMP response element (CRE),was prepared in a similar manner.

GPR7 expression plasmid and pCRE-Luc were transiently expressed in CHOcells, to which the expression vector inserted with no receptor gene wastransfected. The CHO cells were plated on a 96-well plate (CorningCostar, Inc.) in 40,000 cells/well in a medium volume of 100 μl,followed by incubation overnight at 37° C. For incubation on the plate,DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, Inc.) supplementedwith 10% fetal cow serum only was used as medium.

Each plasmid was diluted to a concentration of 240 ng/μl and added to240 μl of Opti-MEM-I (Gibco BRL, Inc.) in a ratio of 9 μl of GPR7expression plasmid to 1 μl of pCRE-Luc. The mixture was mixed with anequal volume of a mixture obtained by adding 10 μl of Lipofectamine 2000(Gibco BRL, Inc.) to 240 μl of Opti-MEM-I (Gibco BRL, Inc.) in a similarmanner to produce the liposome-plasmid complex in accordance with theinstruction manual attached to Lipofectamine 2000. The complex was addedin 25 μl each/well to the culture medium of CHO cells. Four hours later,the culture medium was replaced by an assay buffer (DMEM supplementedwith 0.1% bovine serum albumin) to make the medium serum-free, followedby incubation overnight at 37° C.

Example 5

Expression of Ligand Gene in CHO Cells

The human ligand cDNA-inserted expression plasmid pAK-S64 for animalcells prepared in EXAMPLE 1 was transiently expressed in CHO cells in amanner similar to EXAMPLE 4, except that the cells were plated on a6-well plate (Falcon Corp.) in 600,000 cells/well and incubatedovernight, and then the ligand gene plasmid was introduced. The plasmidwas diluted to a concentration of 240 ng/μl and an aliquot of 10 μl wasadded to 240 μl of Opti-MEM-I. The mixture was mixed with an equalvolume of a mixture obtained in a similar manner by adding 10 μl ofLipofectamine 2000 to 240 μl of Opti-MEM-I to produce theliposome-plasmid complex in accordance with the method described in theinstruction manual attached to Lipofectamine 2000. Each of them wasadded in 500 μl each/well to the culture medium of CHO cells. Four hourslater, the culture medium was replaced by the assay buffer to make themedium serum-free. The medium of each well was recovered 18 hours afterthe medium exchange to obtain the CHO cell culture supernatantcontaining the ligand peptide.

Example 6

Detection of Luciferase Activity Suppression by S64 Expression CellSupernatant in CHO Cells with Transient Expression of GPR7

The pAK-S64 expression culture supernatant prepared in EXAMPLE 5 andforskolin in the final concentration of 2 μM were added to the culturemedium of CHO cells wherein GPR7 was transiently expressed according tothe procedures of EXAMPLE 4. The culture supernatant of CHO cellswherein a ligand gene-free empty expression vector (pAKKO-111H) wastransiently expressed according to the procedures of EXAMPLE 5 was addedas well. At this stage, the expression supernatant was diluted to 2-,4-, 8- and 16-fold with the assay buffer. After the addition of thesupernatant, incubation was carried out for 4 hours at 37° C. to causethe promotion or suppression of transcription/translation of thereporter (luciferase) gene via intracellular signal transduction inducedby the agonist activity of ligand mediated by the receptor. Aftercompletion of the incubation, the assay buffer was removed from eachwell and 50 μl each of PicaGene LT2.0 (Toyo Ink Mfg. Co., Ltd.) as aluminescent substrate was added to the well. After the cells were lysedand thoroughly mixed with the substrate, the luminescence amountassociated with the expression induction level of the reporter gene ineach well was assayed by using a plate reader (ARVOsx Multi-labelCounter, Perkin Elmer, Inc.). As the result, the expression suppressionof the reporter gene was detected as a decreased luciferase activityonly when the culture supernatant of pAK-S64 was added (FIG. 8).Moreover, the degree of this suppression was dependent on theconcentration of the pAK-S64 culture-supernatant. This indicates thatthe product expressed by the plasmid inserted into pAK-S64 transducedthe intracellular signal mediated by GPR7, i.e., acted as a ligand toGPR7.

Example 7

Detection of Luciferase Activity Suppression by S64 Expression CellSupernatant in CHO Cells with Transient Expression of TGR26

The TGR26 expression plasmid DNA was prepared in a manner similar toEXAMPLE 4, using expression plasmid pAKKO-111H for animal cells in whichTGR26 DNA obtained in REFERENCE EXAMPLE 3 was inserted by publicly knownmethods. The plasmid DNA and the luciferase gene were likewise expressedtransiently in the CHO cells according to the procedures of EXAMPLE 4.To the cells, the pAK-S64 expression culture supernatant prepared inEXAMPLE 5 and the culture supernatant of cells wherein the emptyexpression vector alone was expressed, were added and after forskolinwas further added thereto in the final concentration of 2 μM, it wasattempted to detect the ligand activity in a manner similar to EXAMPLE6. As the result, the pAK-S64 supernatant decreasedconcentration-dependently the luciferase activity increased by forskolin(FIG. 9).

Example 8

Suppression of cAMP Production Level in GPR7-Expressed CHO Cells by S64Expression Cell Supernatant

Using the plasmid for GPR7 expression prepared in EXAMPLE 4, CHO-GPR7 asCHO cells capable of stably expressing GPR7 was prepared by publiclyknown methods. Mock CHO cells were plated on a 96-well plate(Beckton-Dickinson, Inc.) in 20,000 cells/well. After incubationovernight at 37° C. under 5% CO₂, the culture medium was used for theassay. The sample buffer used was Dulbecco's modified Eagle's medium(DMEM, Gibco BRL, Inc.) supplemented with 0.1% bovine serum albumin(Sigma) and 0.2 mM IBMX (Sigma). The cells were washed twice with thesample buffer. After pre-incubation at 37° C. for 30 minutes under 5%CO₂, the cells were further washed twice and the sample was addedthereto followed by incubation at 37° C. for 30 minutes under 5% CO₂.The cells were further washed twice and the sample was added thereto at37° C. for 30 minutes. Four kinds of the samples were the sample bufferalone (intact), 1 μM of forskolin (Wako Pure Chemical Industries, Ltd.)as a reagent to stimulate an increase of cAMP production, simultaneousaddition of the CHO cell culture supernatant obtained in EXAMPLE 5prepared by transient expression of pAK-S64 (S64 supernatant) andforskolin, and simultaneous addition of the culture supernatant ofpAKKO-111H-expressed CHO cells (pAKKO supernatant) and forskolin. Afterincubation in the presence of the sample, the intracellular cAMPproduction level was assayed by using cAMP Screen System (ABI). Theresults revealed that the intracellular cAMP production level wasCHO-GPR7-specifically suppressed by adding the S64 supernatant (FIG. 10)and no suppression of the intracellular cAMP production level was notedin the mock CHO cells (FIG. 11).

Example 9

Study of Tissue Distribution of GPR7 Ligand mRNA in Rat by RT-PCR

Various organs were withdrawn from Wistar rat. The total RNA andpoly(A)⁺ RNA were prepared using Isogen (Nippon Gene Co., Ltd.) and mRNApurification kit (Pharmacia), respectively, according to the respectiveinstruction manuals. After 1 μg of poly(A)⁺ RNA was digested with DnaseI(Amplification Grade, Gibco BRL, Inc.), a 160 ng aliquot was treated at42° C. using RNA PCR Kit (TaKaRa Shuzo Co., Ltd.) according to theinstruction manual to synthesize cDNA. The cDNA synthesized was made asolution of 4 ng/μl when calculated as poly(A)⁺ RNA and used as atemplate for RT-PCR thereafter. Using Sequence Detection System Prism7700 (PE Biosystems), RT-PCR was carried out, wherein primers:5′-CTGTCGAGTTTCCACAGGTTCC-3′ (SEQ ID NO:63) and5′-TTGCGCAGAGGTACGGTTCC-3′ (SEQ ID NO:64) were used for amplificationand detection, and 5′-(Fam)-CGTGCCAAGAAACGCGTGACCTTGTT-(Tamra)-3′ (SEQID NO:65) was used as TaqMan probe. In the reaction solution for RT-PCR,0.05 μl each of 100 μM primer solution, 0.5 μl of 5 μM TaqMan probe, 2.5μl of 10× reaction solution attached, 2.5 μl of dNTP (10 mM) and 0.5 μlof the cDNA solution prepared above were added to 12.5 μl of TaqManUniversal PCR Master Mix (PE Biosystems), to which distilled water wasadded to make the total solution volume 25 μl. The reaction solution wasapplied to PCR using Thermal Cycler 9600. After denaturation at 50° C.for 2 minutes and 95° C. for 10 minutes, PCR was carried out byrepeating 40 times the cycle set to include 95° C. for 15 seconds and60° C. for 1 minute. The expression level of GPR7 ligand mRNA in thevarious tissues in rat was assessed in terms of the copy number per 1 ngof poly(A)⁺ RNA (FIG. 12).

Example 10

Purification of Endogenous GPR7 Ligand from Bovine Hypothalamus.

Since it was found that human GPR7 ligand precursor mRNA was abundantlyexpressed in hypothalamus and spinal cord, the endogenous GPR7 ligandwas purified from bovine hypothalamus as the starting material, usinghuman GPR7-expressed CHO cells. The purification was performed using asan indicator the intracellular cAMP production suppressing activity (asdetermined using cAMP-Screen System (ABI)).

First, 1.0 kg of bovine hypothalamus in a frozen state was boiled inMilli-Q Water. After cooling, acetic acid was added to become 1M, whichwas then homogenized with a polytron. After agitation overnight, thehomogenate was centrifuged to give the supernatant. Trifluoroacetic acid(TFA) was added to the supernatant in 0.05% and the mixture was appliedto C18 Column (Prep C18 125 Å; Waters). The peptide bound to the columnwas stepwise eluted with 10%, 40% and 60% acetonitrile containing 0.5%TFA. A 2-fold volume of 20 mM ammonium acetate (pH 4.7) was added to the40% acetonitrile fraction for dilution. The mixture was applied to ionexchange column HiPrep CM-Sepharose FF (Pharmacia). The peptide bound tothe ion exchange column was eluted on a concentration gradient of 0 to0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. A 2-fold volume of cold acetone was added to the NaClfraction (0.3 to 0.35 M) containing the active substance mostabundantly. The precipitates were removed by centrifugation and thesupernatant was concentrated through an evaporator. TFA was added to theconcentrated supernatant in 0.1%. The mixture was applied to reversephase HPLC column RESOURCE RPC (Pharmacia) to effect further separation.The separation from RESOURCE RPC was performed on a concentrationgradient of 20 to 30% acetonitrile, whereby the main activity was elutedon about 22% acetonitrile. A 3-fold volume of cold acetone was added tothe active fraction. The precipitates were removed by centrifugation andthe supernatant was concentrated through the evaporator. TFA was addedto the concentrated supernatant in 0.1%. The mixture was applied toreverse phase HPLC column Vydac C18 218TP5415 (Vydac) to effect furtherseparation. The separation from Vydac C18 218TP5415 was performed on aconcentration gradient of 20 to 30% acetonitrile, whereby the mainactivity was eluted on about 25% acetonitrile. The active fraction wasseparated through cation exchange column TSK-gel CM-2SW (Toso Co., Ltd.)on a concentration gradient of 0.3 to 0.5 M NaCl in 20 mM ammoniumacetate (pH 4.7) containing 10% acetonitrile, whereby the main activitywas eluted on about 0.5 M NaCl. TFA was added to the activity-containingfraction from CM-2SW column in 0.1%. The final purification was madethrough reverse phase HPLC column μRPC C2/C18 SC2.1/10 on aconcentration gradient of 16 to 24% acetonitrile. Thus, a single peakwhich coincided with the activity was obtained (FIG. 13).

Example 11

N-Terminal Amino Acid Sequencing of the Finally Purified Product andDetermination of its Molecular Weight by Mass Spectrum

With respect to the finally purified product obtained in EXAMPLE 10,approximately a half was analyzed with a protein sequencer (model491cLC; Applied Biosystems) for the N-terminal amino acid and the otherhalf was analyzed by ESIMS (Thermoquest).

As a result of the N-terminal sequencing, the sequence corresponding tothe positions 26 to 49 of bovine GPR7 ligand precursor could be read incycles 2 to 25 (FIG. 14). Since cycle 1 could not be identified (x), theproduct was presumed to undergo a post-translational modification. Thesequence after cycle 2 was identified clearly to be the sequencedescribed above.

In ESIMS (FIG. 15, upper column), the value of 3241.5 was obtained in afull mass scan mode. Based on the molecular weight calculated from themass spectrum and the analysis results of MS/MS spectrum (FIG. 15, lowercolumn), it was presumed that either one of the N-terminal two residueswould undergo a post-translational modification. Taking into account theN-terminal sequencing results together, it was presumed that Trp atposition 1 would be modified.

Putting the isotonic profile (FIG. 16) of trivalent molecular ionsdetermined in a zoom scan mode together, the substance was presumed tobe a GPR7 ligand of 29 residues, which would be brominated on thetryptophan residue at the 1-position.

To confirm the presumption, PTH standard was prepared fromDL-5-bromotryptophan (Aldrich) and DL-6-bromotryptophan (Biosynth),followed by sequencing.

To 200 nmols of DL-5-bromotryptophan or DL-6-bromotryptophan, 20 μl ofethanol:triethylamine:DW:phenyl isothiocyanate (sigma)=7:1:1:1 wasadded. The mixture was reacted at room temperature for 20 minutes. Afterdrying, 50 μl of TFA was added thereto. The mixture was reacted at 50°C. for 10 minutes. After during, 50 μl of HCl: methanol=1:1 was addedthereto, followed by reacting at 50° C. for 10 minutes. The reactionmixture was purified on reverse phase HPLC to give the PTH derivative of5-bromotryptophan or 6-bromotryptophan. The final product was identifiedon a protein sequencer (FIGS. 17 and 18). These PTH derivatives weremixed with 20 amino acid PTH standard (ABI), and a protocol was preparedto separate the derivatives (TABLES 1 and 2). When the endogenous bovineGPR7 ligand was analyzed, the amino acid at the 1-position coincidedwith the peak of PTH-6-bromotryptophan (FIG. 19).

Based on the results of analysis, the finally purified product frombovine hypothalamus was found to be a peptide of 29 amino acids (SEQ IDNO:67) corresponding to the 25th Trp to. 53rd Ala of bovine GPR7 ligandprecursor, in which Trp at position 1 was 6-brominated by apost-translational modification.

TABLE 1 Cycle # Cartridge cycle Flask cycle Gradient Pulsed-Liquid cLC:Default Cart-PL 6mmGFF cLC Flask Normal cLC Normal 1 cLC 1 None PreparePump cLC Prepare Pump cLC 2 None Flask Blank cLC Normal 1 cLC 3 CartBegin cLC Flask Standard cLC Normal 1 cLC BrTrp-liquid cLC: DefaultCart-PL 6mmGFF cLC Flask Normal cLC Normal for BrW cLC 1 Sample washPrepare Pump cLC Prepare Pump cLC 2 None Flask Blank cLC BrTrp cLC 3Cart Begin cLC Flask Standard cLC BrTrp cLC 4 Cart-PL 6mmGFF cLC FlaskNormal cLC BrTrp cLC 5 Cart-PL 6mmGFF cLC Flask Normal cLC BrTrp cLC

TABLE 1 shows a comparison in cycle and gradient between a method fornormal peptide (Pulsed-Liquid cLC) and a method for bromotryptophan(BrTrp-liquid cLC).

TABLE 2 Time (min) 0 0.4 4 22 22.6 29 33 Normal 1 % B 8 10 20 47 90 9070 cLC Normal % B 8 10 20 44 90 90 70 for BrW cLC 0.0 0.4 4 22.0 28.028.6 32.0 33.0 BrTrp cLC % B 8 10 20 44 44 90 90 70

TABLE 2 shows a comparison in gradients prepared for the analysis of anormal peptide (Normal 1 cLC) and bromotryptophan (Normal for BrW cLC,BrTrp cLC), on 491cLC protein sequencer (ABI).

The N-terminal sequencing was performed on 491cLC protein sequencer(ABI) by the analysis method for normal peptide (Normal 1cLC) with amodification (BrTrp-liquid cLC) for bromotryptophan analysis. Otherconditions than those described above were set as instructed in themanual provided by the manufacturer. When a modified gradient (BrTrpcLC) is used, 5- and 6-bromotryptophans, which have different positionsfor Br added, can be discriminated from each other.

When the analysis is made by the modified method or BrTrp-liquid cLC,the gradient for analysis of 5-/6-bromotryptophan is adapted only toblank, standard and up to cycle 2, and a different gradient (Normal forBrW cLC) is adapted to and after cycle 3.

Example 12

Acquisition of Bovine GPR7 Ligand Precursor Gene from BovineHypothalamus cDNA by PCR

Using bovine hypothalamus cDNA as a template, PCR was performed foramplification, using two synthetic DNAs below.

(SEQ ID NO:82) BF1: 5′-CCCATGGCCGGGCCCGCGATGCTGGTCGCC-3′ (SEQ ID NO:83)BR1: 5′-TCACTTGCGACAGTCCGAGGCGCTGAGCGA-3″

The reaction solution for PCR contained 1 μl of cDNA solution, 0.5 μl ofBF1 (10 μM), 0.5 μl of BF2 (10 μM), 2.5 μl of 10× reaction solutionattached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq (CLONTECH, Inc.),to which 17.5 μl of Otsuka distilled water was added to make the totalvolume 25 μl. The reaction solution was applied to PCR using ThermalCycler 9600. The conditions for PCR were set forth: after denaturationat 95° C. for 2 minutes, the cycle set to include 98° C. for 10 seconds,60° C. for 20 seconds and 72° C. for 20 seconds was repeated 35 times.After it was confirmed by electrophoresis using an aliquot of the PCRproduct that the PCR product of about 400 bp was amplified, the PCRproduct was purified using Quiagen PCR purification Kit and directlysequenced to obtain the sequence shown by FIG. 20. The amino acidsequence deduced from the DNA sequence of FIG. 20 was the sequence shownin FIG. 21. Next, the PCR product recovered from the gel was subclonedto Escherichia coli JM109 using TA Cloning Kit (Invitrogen, Inc.) toacquire Escherichia coli JM109/pTAbGPR7L-1. Plasmid pTAbGPR7L-1 wasextracted from Escherichia coli obtained by the subcloning, using aplasmid extractor (Kurabo Co., Ltd.) to identify the base sequence ofthe inserted fragment. It was confirmed that the sequence was the sameas that of bovine GPR7 ligand cDNA.

Example 13

Synthesis of GPR7 Ligand

GPR7 ligand (GPR7L) and GPR8 ligand (GPR8L) were synthesized by theFmoc/DCC/HOBt protocol, using ABI 433 peptide synthesizer.DL-6-Bromotryptophan (Biosynth) was changed toBoc-DL-6-bromotryptophan-OMe and then subjected to chiral resolution,which was used for peptide synthesis, respectively.

(1) DTrp (6Br)1-human GPR7L (29) (wherein N-terminal D-tryptophan in theamino acid sequence represented by SEQ ID NO: 4 was brominated at the6-position):(D-Trp(6Br)-Tyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu-Arg-Arg-Ser-Pro-Tyr-Ala)(SEQ ID NO: 107)(2) LTrp(6Br)1-human GPR7L (29) (wherein N-terminal L-tryptophanin theamino acid sequence represented by SEQ ID NO:4):

-   (Trp(6Br)-Try-Lyr-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Gly-Leu-Leu-Ser-Gly-Arg-Arg-Ser-Pro-Try-Ala)    (3)Trp1-human GPR7L (29) (SEQ ID NO:4):-   (Trp-Try-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Arg-Arg-Ser-Pro-Try-Ala)    (4) Trp1-human GPR7L (23) (SEQ ID NO:1):-   (Trp-Try-Lys-Pro-Ala-Ala-Gly-His-Ser-Ser-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Leu)    (5) DTrp(6Br)1-bovine GPR7L (29) (wherein N-terminal D-tryptophan in    the amino acid sequence represented by SEQ ID NO: 67 was brominated    at the 6-position):    (D-Trp(6Br)-Tyr-Lys-Pro-Thr-Ala-Gly-Gln-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Tyr-Ala)    (SEQ ID NO: 108)    (6) LTrp(6Br)1-bovine GPR7L (29) (wherein N-terminal L-tryptophanin    the amino acid sequence represented by SEQ ID NO:67 was brominated    at the 6-position):-   (Trp(6Br)-Tyr-Lys-Pro-Thr-Ala-Gly-Gin-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Try-Ala)    (7) Trp1-bovine GPR7L (29) SEQ ID NO:67):-   (Trp-Tyr-Lys-Pro-Thr-Ala-Gly-Gin-Gly-Tyr-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Gly-Phe-His-Arg-Ser-Pro-Try-Ala)    (8) Trp1-rat GPR7L(29)(SEQ ID NO:67):-   (Trp-Tyr-Lys-Pro-Ala-Ala-Gly-Ser-HisI-His-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Ser-Phe-His-Arg-Phe-Pro-Ser-Thr)    (9) Trp1-rat GPR7L (24) (SEQ ID NO:3):-   (Trp-Try-Lys-Pro-Ala-Ala-Gly-Ser-His-His-Tyr-Ser-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Ser-Ser-Phe-His)    (10) Trp1-human GPR8L (23) (SEQ ID NO:100):-   (Trp-Try-Lys-His-Val-Ala-Ser-Pro-Arg-Tyr-His-Thr-Val-Gly-Arg-Ala-Ala-Gly-Leu-Leu-Met-Gly-Leu)    (WO 01/98494)

Example 14

Effect of GPR7 Ligand on Feed Uptake in Rat by Lateral VentricularInjection

The effect of GPR7 ligand (GPR7L) on feed uptake in rat by lateralventricular injection was examined. Rat was caged at room temperature of25° C. while lighting for 8 to 20 o'clock. Mature Wistar male rats(300-320 g body weight upon surgery) were anesthetized with anintraperitoneal injection of 50 mg/kg pentobarbital and placed in a ratbrain stereotaxic instrument. The level of incisor bar was 3.3 mm belowthe interaural line. The skull was exposed, and using a dental drill ahole was made on the bone for implantation of guide cannula AG-8 (innerdiameter of 0.4 mm, outer diameter of 0.5 mm, EICOM Corporation). Inaddition, an anchor screw was buried at 3 positions around the hole. Astainless-steel guide cannula, AG-8, was inserted in such a manner thatthe tip was situated at the upper part of the lateral ventricle.Stereotaxic coordinates were taken from the atlas according to the atlasof Paxinos & Watson (1986) from bregma AP: −0.8 mm, L:1.5 mm and H:−4.5mm. The guide cannula was secured to the skull using a dental cement andan anchor screw. A stainless-steel dummy cannula AD-8 (outer diameter of0.35 mm, EICOM Corporation) was then passed through the guide cannulaand locked in position with a cap nut (EICOM Corporation). After thesurgery, rats were housed in individual cages.

After the guide cannula was implanted, rats were caged for about a weekto recover from surgical operation. The cap nut and dummy cannulainserted into the rat skull were disconnected and instead, astainless-steel microinjection cannula AMI-9 (inner diameter of 0.17 mm,outer diameter of 0.35 mm, EICOM Corporation) connected to a Teflon(registered trademark) tube (length of 50 cm, inner diameter of 0.1 mm,outer diameter of 0.35 mm, EICOM Corporation) was inserted into theskull. The length of the microinjection cannula was adjusted beforehandto expose the tip from the guide cannula by 1 mm. One end of the Teflon(registered trademark) tube was connected to a microsyringe pump andeither sterile distilled water (Otsuka Pharmaceutical Co., Ltd.) ornon-brominated GPR7L (Trp1-bovine GPR7L (29), SEQ ID NO:67) synthesizedin EXAMPLE 13 dissolved in distilled-water was infused, in a totalvolume of 10 μl (10 nmols/rat), into the lateral ventricle at a flowrate of 5 μl/min. After a 2 minute standby time following the infusion,the microinjection cannula was disconnected and the dummy cannula waslocked in position again with a cap nut. The infusion was continued from19:00 to 20:00 o'clock, and the feed uptake thereafter was measured withpassage of time, using a feed uptake measuring device Feed-Scale(Columbus, Inc.). As shown in FIG. 22, a significant increase in feeduptake from 2 hours after the administration was noted in the groupadministered with non-brominated GPR7L, as compared to the controlgroup.

Example 15

Assay for Bovine Endogenous GPR7 Ligand by FTMS

Bovine endogenous GPR7 was analyzed on Apex II (Burker Daltonics) byESIFTMS (FIG. 23, upper). In FIG. 23, [M+5H]⁵⁺ ions are shown in anenlarged drawing and the [M+5H]⁵⁺ ion isotope theoretical profile of1Br-added bovine GPR7L is shown at the lower column. The modifiedproduct was identified to be Br, since the isotope profile and massspectral data matched well.

Example 16

Study of Tissue Distribution of GPR7 Ligand mRNA in Human by RT-PCR

The expression level of mRNA was assayed in a manner similar to EXAMPLE9, except that cDNAs used as templates were prepared from polyA+ RNA(CLONTECH) derived from various human organs by the followingprocedures. Using reverse transcriptase or SuperScript II as a randomprimer and reverse transcriptase, cDNA was prepared from 1 μg of RNA andthe reaction was carried out at 42° C. in accordance with theinstruction manual attached. After completion of the reaction, theprecipitates in ethanol were dissolved in 100 μl. Also, the expressionlevel was quantified in a manner similar to EXAMPLE 9 using SequenceDetection System Prism 7700, except that the following were used foramplification and detection: 5′-CGCTCCCAGCCCTACAGA-3′ (SEQ ID NO:90) and5′-TCGCCTTGCACTGGTAGGTC-3′ (SEQ ID NO:91) as primers and as TaqManprobe, 5′-(Fam) AGCCTCGCTGTGTGCGTCCAGGAC-(Tamra)-3′ (SEQ ID NO:92).

The expression level of GPR7 mRNA in various human tissues obtained wasassessed in terms of a copy number per 1 ng of poly(A)⁺ RNA (FIG. 24).

Example 17

Study of Tissue Distribution of Rat GPR7 (rat TGR26) mRNA by RT-PCR

The expression level of mRNA was assayed in a manner similar to EXAMPLE9. Using the cDNAs derived from various organs of rat used in EXAMPLE 9,the expression level of rat GPR7 mRNA was determined, except that exceptthat the following were used for amplification and detection:5′-TGCGTGCTATCCAGCTAGACAG-3′ (SEQ ID NO:93) and5′-AGAGGAGGCACACAGCCAGAAT-3′ (SEQ ID NO:94) as primers and5′-(Fam)CGTGCCAAGAAACGCGTGACCTTGTT-(Tamra)-3′ (SEQ ID NO:95) as TaqManprobe.

The expression level of GPR7 mRNA in various tissues of rat obtained wasassessed in terms of a copy number per 1 ng of poly(A)⁺ RNA (FIG. 25).

Example 18

Acquisition of Bovine GPR7 Gene from Bovine Hypothalamus cDNA by PCR

Using bovine hypothalamus cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs.

(SEQ ID NO:96) BGPR7F: 5′-GTCGACCGAGTGTCTGTCCTCGCCAGGATG-3′ (SEQ IDNO:97) BGPR7R: 5′-GCTAGCTCCTTGTTATCGGGCTCAGGAGGTGGT-3′

The reaction solution for PCR contained 1 μl of cDNA solution, 0.5 μl ofBGPR7F (10 μM), 0.5 μl of BGPR7R (10 μM), 2.5 μl of 10× reactionsolution attached, 2.5 μl of dNTP (10 mM) and 0.5 μl of KlenTaq(CLONTECH, Inc.), to which 17.5 μl of Otsuka distilled water was addedto make the total volume 25 μl. The reaction solution was applied to PCRusing Thermal Cycler 9600. The conditions for PCR were set forth: afterdenaturation at 95° C. for 2 minutes, the cycle set to include 98° C.for 10 seconds, 60° C. for 20 seconds and 72° C. for 60 seconds wasrepeated 40 times. It was confirmed by electrophoresis using an aliquotof the PCR product that the PCR product of about 1000 bp was amplified.The PCR product was then purified using Quiagen PCR purification Kit(QIAGEN, Inc.) and directly sequenced to obtain the sequence shown byFIG. 26. The amino acid sequence deduced from the DNA sequence of FIG.26 was the sequence shown in FIG. 27. Next, the PCR product recoveredfrom the gel was subcloned to Escherichia coli JM109, using TA CloningKit (Invitrogen, Inc.), to acquire Escherichia coli JM109/pTAbGPR7.Plasmid pTAbGPR7 was extracted from Escherichia coli obtained by thesubcloning, using a plasmid extractor (Kurabo Co., Ltd.) to identify thebase sequence of the inserted fragment. It was confirmed that thesequence was the same as that of bovine GPR7 receptor cDNA.

Example 19

Acquisition of Bovine GPR8 Gene from Bovine Hypothalamus cDNA by PCR

Using bovine hypothalamus cDNA as a template, amplification wasperformed by PCR using the following two synthetic DNAs.

(SEQ ID NO:98) BGPR8F: 5′-GTCGACCATGATGGAGGCCACTGGGCTGGAAGG-3′ (SEQ IDNO:99) BGPR8R: 5′-GCTAGCTTATGCCCCCTGGCACCGACATGCGGT-3′

PCR was carried out in a manner similar to EXAMPLE 18. The PCR productobtained was purified using Quiagen PCR purification Kit and directlysequenced to obtain the sequence shown by FIG. 28. The amino acidsequence deduced from the DNA sequence of FIG. 28 was the sequence shownin FIG. 29. Next, the PCR product recovered from the gel was subclonedto Escherichia coli JM109, using TA Cloning Kit (Invitrogen, Inc.), toacquire Escherichia coli JM109/pTAbGPR8. Plasmid pTAbGPR8 was extractedfrom Escherichia coli obtained by the subcloning, using a plasmidextractor to identify the base sequence of the inserted fragment. It wasconfirmed that the sequence was the same as that of bovine GPR8 cDNA.

Example 20

Purification of GPR7 Ligand from the Culture Supernatant of Human GPR7Ligand-Expressed CHO Cells

The culture supernatant of human GPR7 ligand-expressed CHO cellsconstructed in EXAMPLE 5 was collected to make the volume 2 liters andstored at −80° C. The culture supernatant was thawed, boiled in hotwater and then centrifuged to obtain the supernatant. Trifluoroaceticacid (TFA) was added in 0.05% to the supernatant and the mixture wasapplied to C18 Column (Prep C18 125 Å; Waters, Inc.). The peptide boundto the column was stepwise eluted with 10, 40 and 60% acetonitrilecontaining 0.5% TFA. The 30% acetonitrile fraction was diluted with a3-fold volume of 20 mM ammonium acetate (pH 4.7) and the dilution wasapplied to ion exchange column HiPrep CM-Sepharose FF (Pharmacia). Thepeptide bound to the column was eluted in a concentration gradient of 0M to 0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. An aliquot of each fraction was desalted using Sep-Pakplus C18 Cartridge (Waters, Inc.), the intracellular cAMP productionsuppression activity specific to human GPR7-expressed CHO cells wasassayed. TFA was added in 0.1% to the CM-Sepharose fraction found tohave a specific activity to human GPR7-expressed CHO cells, which wasseparated by passing through reverse phase HPLC column RESOURCE RPC(Pharmacia). The separation through RESOURCE RPC was carried out in aconcentration gradient of 15-30% acetonitrile. The main intracellularcAMP production suppression activity specific to the humanGPR7-expressed CHO cells was eluted on about 22% acetonitrile. Thisactive fraction was separated by passing through cationic ion exchangecolumn TSK gel CM-SW (Toso Co., Ltd.) in a concentration gradient of0.2-0.5 M NaCl in 20 mM ammonium acetate (pH 4.7) containing 10%acetonitrile. The main intracellular cAMP production suppressionactivity was eluted on about 0.3 M NaCl. TFA was added in 0.1% to thefraction from the CM-2SW column. Final purification of the mixture onreverse phase column μRPC C2/C18 SC2.1/10 gave a single peak, whichcoincided with the intracellular cAMP production suppression activityspecific to the human GPR7-expressed CHO cells (FIG. 30).

Analysis of the N-terminal amino acids in the finally purified productusing a protein sequencer (model 492; Applied Biosystems, Inc.) gave theamino acid sequence shown in FIG. 31.

Also, the molecular weight of the finally purified product wasdetermined using ESI-MS (Thermoquest, Inc.) and found to be 2505.6 (FIG.32).

Based on these analytical results, the finally purified product wasfound to be a peptide of 24 amino acids corresponding to Trp25 to Arg48in the precursor.

Example 21

Preparation of Iodine-Labeled Human GPR7 Ligand

A mixture of 20 μl of hGPR7L-23 (SEQ ID NO:1) (0.1 mM or 1 mM), 20 μl oflactoperoxidase (Sigma; prepared using 10 μg/ml and 0.1M HEPES-NaOHpH7.0), 20 μl of Idoine-125 (manufactured by Amersham, MS-30, 74 MBq),20 μl of 0.005% hydrogen peroxide (manufactured by Wako Pure ChemicalIndustries, Ltd.) was allowed to stand at room temperature for 20 to 30minutes. Then, 600 μl of 0.1% TFA was added to the mixture. The mixturewas applied to reverse phase HPLC for separation and the peaks of thetwo'labeled products were fractionated in a tube charged with 1 mL ofDMSO. Immediately, the fraction was stored on ice and an aliquot wasprovided for measurement of the radioactivity with a γ-counter. Theremaining preparation was dispensed and stored at −30° C.

Example 22

Preparation of Human GPR7-Expressed CHO Cell Membrane Fraction

Human GPR7-expressed CHO cells were cultured in a flask. The flask waswashed with 5 mM EDTA/PBS to strip the cells off. The cells werestripped off with 5 mM EDTA/PBS and centrifuged for recovery. Therecovered cells were suspended in 25 mL of a buffer for preparingmembrane fraction (50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.1% bovine serumalbumin (manufactured by Sigma), 0.5 mM PMSF (manufactured by Wako PureChemical Industries, Ltd.), 20 μg/mL leupeptin (manufactured by PeptideResearch Institute), 0.1 μg/mL pepstatin A (manufactured by PeptideResearch Institute) and 4 μg/mL E-64 (manufactured by Peptide ResearchInstitute)), followed by homogenization on ice using a polytron (12,000rpm, 15 seconds×3 times). The homogenate was centrifuged at 4° C. under1,000 g for 10 minutes with a high speed cooling centrifuge to recoverthe supernatant. After 25 mL of the buffer for preparing membranefraction was added to the precipitates, the supernatant was recovered bythe same procedures. These supernatants were combined, applied to a cellstrainer, dispensed in a super centrifuge tube and centrifuged at 4° C.under 100,000 g for an hour. The pellets were recovered and suspended ina small quantity of the buffer for preparing membrane fractions. Afterfurther suspending with a Teflon (registered trademark) homogenizer, analiquot of the suspension was used to determine a protein level. Theremaining suspension was dispensed and stored at −80° C.

Example 23

Scatchard Analysis Using Human GPR7-Expressed CHO Cell Membrane Fraction

Scatchard analysis was performed using human GPR7-expressed CHO cellmembrane fraction and [Tyr (¹²⁵I)¹¹]-hGPR7L-23 (SEQ ID NO:1). Themembrane fraction was diluted in a final concentration of 1 μg/well withan assay buffer (50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.1% bovine serumalbumin (manufactured by Sigma), 0.5 mM PMSF (manufactured by Wako PureChemical Industries, Ltd.), 20 μg/mL leupeptin (manufactured by PeptideResearch Institute), 0.1 μg/mL pepstatin A (manufactured by PeptideResearch Institute) and 4 μg/mL E-64 (manufactured by Peptide ResearchInstitute)), and the labeled product was diluted in 400 μM, 200 μM, 100μM, 50 μM, 25 μM and 10 μM. Using a polyproprene-made 96-well plate, 50μl each of the assay buffer alone (total) and hGPR7L-23 (NSB) in a finalconcentration of 2 μM were dispensed in each well. To each well, 25 μlof the solution of the labeled product was added. After agitation, 25 μlof the diluted membrane fraction was added/mixed, followed by incubationat room temperature for 1.5 hour. Using a cell harvester for the 96-wellplate, adsorption was made onto a filter unit (GF/C, treated withpolyethyleneimine), which had previously been made wet with an assaybuffer (50 mM Tris-HCl, pH7.5). After washing 5 times with the assaybuffer, the filter unit was thoroughly dried. As an input, the dilutionof the labeled product was directly added to a filter unit (GF/C,treated with polyethyleneimine) and dried. After 50 μl of a liquidscintillator was dispensed thereto, the radioactivity was counted on aTop Count (Packard) and the data was analyzed in triplet (FIG. 33),thereby to obtain the values of Bmax=1.28 pmol/mg protein and Kd=35.5μM.

Example 24

Test on Binding Inhibition of Various Peptides Against HumanGPR7-Expressed CHO Cells

Using the assay buffer, the human GPR7-expressed CHO cell membranefraction was diluted in a final concentration of 1 μg/well andiodine-labeled hGPR7L-23 (SEQ ID NO:1) was diluted in a finalconcentration of 100 μM. The peptides shown in TABLE 3 are thoseobtained by diluting the stock solution of 10 M or 10⁻³ M with the assaybuffer in 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M and 10⁻¹¹ M.As NBS, hGPR7L-23 was prepared in a final concentration of 10⁻⁵ M. Thesample solution and NSB prepared were dispensed on a polypropylene-made96-well plate, the assay buffer was dispensed thereto to make the totalvolume 50 μl, and 25 μl of the iodine-labeled product dilution was addedthereto. After agitation, 25 μl of the solution of human GPR7-expressedCHO cell membrane fraction were dispensed thereto and agitated, followedby incubation at room temperature for 1.5 hour. Using a cell harvesterfor the 96-well plate, the culture medium was adsorbed onto a filterunit, which had previously been wetted with an assay buffer (50 mMTris-HCl, pH7.5). After washing 5 times with the assay buffer, thefilter unit was thoroughly dried. After 50 μl of a liquid scintillatorwas dispensed thereto, the radioactivity was counted on a Top Count(Packard) and the data was analyzed in triplet (FIG. 33). The resultsobtained by the test on binding inhibition of various peptides againsthuman GPR7-expressed CHO cell membrane fraction are shown in terms ofIC₅₀ values in TABLE 3.

TABLE 3 Peptide IC50 (nM) DTrp(6Br)1-human GPR7L(29) 13 LTrp(6Br)1-humanGPR7L(29) 0.32 Trp1-human GPR7L(29) 0.33 Trp1-human GPR7L(23) 1.6Trp1-human GPR8L(23) 0.4 DTrp(6Br)1-bovine GPR7L(29) 6.1LTrp(6Br)1-bovine GPR7L(29) 0.34 Trp1-bovine GPR7L(29) 0.31 Trp1-ratGPR7L(29) 0.34 Trp1-rat GPR7L(24) 0.30

Example 25

Comparison in Agonist Activity of Various Peptides on GPR7 andGPR8-Expressed CHO Cells

The intracellular cAMP production suppression activity of peptidesassociated with various GPR7 ligands on CHO cells, in which human GPR7,bovine GPR8, human GPR8, human GPR8, bovine GPR8 and rat GPR7 wereexpressed, was examined. Each of the receptor-expressed cells waspassaged on a 96-well plate in 4×10⁴ cells/well, followed by incubationfor 1 day at 37° C. under 5% CO₂ and 95% air. An assay buffer wasprepared by adding 20 mM HEPES, pH7.4, 0.1% bovine serum albumin and 0.2mM 3-isobutyl-1-methylxanthine (Sigma) to Hanks' Balanced Salt Solution(Gibco BRL). The plate incubated overnight was first washed twice with150 μl of the assay buffer, and then exchanged with 150 μl of the assaybuffer, followed by incubation for 30 minutes at 37° C. in 100% air. Byadding 4 μM forskolin to the assay buffer, a buffer for sample dilutionwas prepared and the stock solution (10⁻² M of 10⁻³ M) was diluted withthe buffer thus obtained to prepare sample solutions in finalconcentrations of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M and 10⁻¹⁰ M. The plateincubated with the assay buffer for 30 minutes was taken out. Afterwashing twice with the assay buffer, 50 μl of the assay buffer and then50 μl of the sample solution were added thereto. Each sample was assayedin triplet. Furthermore, the assay buffer of the same volume forassaying the basal level and a buffer supplemented with forskolin forassaying the maximum level were added. After the plate was incubated for30 minutes at 37° C. in 100% air, the intracellular cAMP level wasassayed using cAMP-Screen™ System (ABI, Inc.) according to the protocolattached to the kit. A difference between the maximum cAMP level and thecAMP level when each sample was added was calculated and the percentageof cAMP production level promoted by forskolin was worked out, which wasmade the cAMP production inhibition rate. The IC₅₀ values of respectivesamples are shown in TABLE 4.

TABLE 4 IC50 (nM)

human GPR7 human GPR8 bovine GPR7 bovine GPR8 rat GPR7 DTrp(6Br)1-humanGPR7L(29) 106 770 360 424 49 LTrp(6Br)1-human GPR7L(29) 0.44 32 1.8 510.47 Trp1-human GPR7L(29) 0.45 49 2.6 52 0.29 Trp1-human GPR7L(23) 0.5844 3.5 47 0.28 Trp1-human GPR8L(23) 0.82 3.7 2.4 4.8 0.32DTrp(6Br)1-bovine GPR7L(29) 35 198 77 34 2.5 LTrp(6Br)1-bovine GPR7L(29)0.58 9.7 1.9 4.0 0.21 Trp1-bovine GPR7L(29) 0.43 8.1 1.2 5.4 0.25Trp1-rat GPR7L(29) 0.86 8.8 1.2 14 0.30 Trp1-rat GPR7L(24) 0.31 2.8 0.513.5 0.18

Example 9

Study of Expression Distribution of GPR7 Ligand mRNA in Rat Brain by InSitu Hybridization

Wistar rat was laparotomized under anesthesia with Nembutal and 250 mlof 0.9% aqueous sodium chloride solution was infused through the leftventricle and then with 250 ml of 4% p-formaldehyde solution. After thebrain withdrawn was immersed in the solution for 4 hours at 4° C., thesolution was replaced by 20% sucrose solution. The brain was immersedfor further 3 days at 4° C. to obtain the brain sample provided foranalysis.

GPR7 ligand, antisense and sense probe were prepared by the followingmethod.

First, rat GPR7 ligand cDNA was inserted into plasmid vector pBluescriptII KS+ (Stratagene) by publicly known methods. This plasmid wasinactivated by treating with restriction enzyme BamHI or XbaI, which wasdissolved in TE, respectively, in 0.52 μg/ml and 0.47 μg/ml. To 2 μl ofthe BamHI-treated product, 40 U of T3 RNA polymerase (Roche), 2 μl ofthe supplied 10× buffer, 20U of RNase inhibitor (Roche) and 2 μl of DIGRNA Labeling Mix, 10× (Roche), water was added to make the final volume20 μl. After the mixture was reacted at 37° C. for 2 hours, the reactionwas terminated by adding 2 μl of 0.2 M EDTA, and the riboprobe formed byethanol precipitation was recovered and used as an antisense probe.Also, to 2 μl of the XbaI-treated product, 40 U of T3 RNA polymerase(Roche), 2 μl of the supplied 10× buffer, 20U of RNase inhibitor (Roche)and 2 μl of DIG RNA Labelling Mix, 10× (Roche), water was added to makethe final volume 20 μl. The mixture was reacted at 37° C. for 2 hours.After the reaction was terminated by adding 2 μl of 0.2 M EDTA, theriboprobe formed by ethanol precipitation was recovered and used as asense probe. The respective concentrations were measured and the probeswere dissolved in RNase free water to set the concentrations ofantisense and sense probes at 0.29 μg/ml and 0.27 μg/ml, respectively.

In situ hybridization was carried out by the following method. The brainsample prepared as described above was sliced on Cryostat CM3050 (Leica)at 25 μm thick in the frontal plane. The slice formed was washed twicewith 10 ml of 4×SSC for 5 minutes. Then, Protenase K (Sigma) was addedto 10 ml of PK buffer (pH7.4, 10 mM Tris-HCl, 10 mM EDTA) in a finalconcentration of 2.5 mg/ml, followed by reacting them at 37° C. for 10minutes. The reaction mixture was washed twice with 10 ml of 4×SSC for 5minutes. Acetic anhydride was added to 10 ml of an acetylation buffer(pH 7.5, 100 mM triethanolamine) in 0.25%, followed by reacting at roomtemperature for 10 minutes. The mixture was washed with 10 ml of 4×SSCfor 5 minutes. After this operation was repeated twice, the slice wasadded to 1 ml of a hybrid buffer (pH 7.4, 60% formamide, 10 mM Tris-HCl,200 μg/ml yeast t-RNA, 1× Denhardt's reagent, 10% dextran sulfate, 600mM NaCl, 0.25% SDS, 1 mM EDTA) supplemented with 0.2 μg/ml each of theantisense and the sense probe. Hybridization was performed at 55° C. for13 hours, followed by washing at 55° C. for 15 minutes twice with 10 mlof a wash buffer (2×SSC, 50% formamide). RNaseA (Sigma) was added toRNase buffer (pH8.0, 10 mM Tris-HCl, 1 mM EDTA, 0.5M NaCl) in 2.5 μg/mland the slice was transferred thereto followed by reacting them at 37°C. for 30 minutes. The reaction mixture was washed at 55° C. for 15minutes with 10 ml of the wash buffer. After this operation was repeatedtwice, the mixture was washed with 0.4×SSC at 55° C. for 15 minutes. Theslice was transferred to a solution mixture of 0.1 g of a blockingsolution (Roche) and Buffer A (pH 7.5, 100 mM Tris-HCl, 150 mM NaCl) toreact them at room temperature for an hour. The slice was transferred to1 ml of Buffer A containing Triton X-100 and supplemented with 0;75U ofanti-digoxygenin-AP, Fab fragments (Roche), followed by reacting them at4° C. for 16 hours. The reaction mixture was then washed with 10 ml ofBuffer A at room temperature for 15 minutes. After this operation wasrepeated twice, the mixture was washed with 10 ml of Buffer B (pH 9.5,100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl₂) at room temperature for15 minutes. To 10 ml of Buffer B, 40 oil of NBT solution (Roche) and 30μl of X-phosphate solution were added, and the slice was transferred tothe mixture to perform a color-forming reaction. After reacting at roomtemperature for 24 hours, the slice was transferred to 50 ml of TE. Theslice was applied onto MAS-coated slide glass (Matsunami Glass Ind.Ltd.). After air-drying, a cover glass was applied thereon with asealant (50% glycerol, 5% gelatin). The areas where the color wasdeveloped specifically to the antisense probe were the medial andlateral preoptic areas of hypothalamus, the lateral hypothalamic field,the CA1-CA3 areas of hippocampal pyramidal cells, the mesencephalicaqueduct ventral division of the midbrain, etc. In these areas, anycolor formation was not detected by the sense probe.

INDUSTRIAL APPLICABILITY

The peptide of the present invention and its DNA, the bovine GPR7 of thepresent invention and its DNA, as well as the bovine GPR8 of the presentinvention and its DNA are useful as pharmaceuticals for theprevention/treatment of, e.g., anorexia, appetite (eating) stimulants,etc.

In addition, the peptide, etc. of the present invention are also usefulfor screening GPR7 agonists or antagonists, etc.

1. An isolated peptide comprising the amino acid sequence of SEQ IDNO:4, which may optionally be brominated at the N-terminal amino acidresidue, or its amide or ester, or a salt thereof.
 2. The peptide or itsamide or ester,or a salt thereof, according to claim 1, which comprisesthe amino acid sequence of SEQ ID NO:4.
 3. The peptide or its amide orester, or a salt thereof, according to claim 1, which consists of theamino acid sequence of SEQ ID NO:4.
 4. The peptide or its amide orester, or a salt thereof, according to claim 1, which consists of theamino acid sequence of SEQ ID NO:4 and which is 6-brominated at theN-terminal tryptophan residue.
 5. An isolated peptide or its amide orester, or a salt thereof, which comprises the amino acid sequence of SEQID NO:19.
 6. An isolated peptide or its amide or ester, or a saltthereof, which comprises the amino acid sequence of SEQ ID NO:22.